Bioanalytical reagent, method for production thereof, sensor platforms and detection methods based on use of said bioanalytical reagent

ABSTRACT

The invention relates to various embodiments of a bioanalytical reagent with at least one vesicle, generated from a living cell, comprising at least one receptor, characterized in that a mechanism of signal transduction triggered by said receptor in the cell used for vesicle generation is preserved in said vesicle, as a component of the bioanalytical reagent. The invention further relates to methods for production of the bioanalytical reagent according to the invention, to bioanalytical detection methods based on the application of said reagent, and to the use of said detection method and of the bioanalytical reagent.

The invention relates to various embodiments of a bioanalytical reagentwith at least one vesicle, generated from a living cell, comprising atleast one receptor, characterized in that a mechanism of signaltransduction triggered by said receptor in the cell used for vesiclegeneration is preserved in said vesicle, as a component of thebioanalytical reagent. The invention further relates to methods forproduction of the bioanalytical reagent according to the invention, tobioanalytical detection methods based on the application of saidreagent, and to the use of said detection method and of thebioanalytical reagent.

Living organisms can perceive an respond to a variety of externalsignals (light, hormones, odours, etc.). These signals are received andprocessed at cell surfaces (=plasma membrane receptors) by receptorswhich transmit the signals through the membrane and trigger a multitudeof processes in the cell interior resulting in a cellular response.

Many plasma membrane receptors are important target molecules oftherapeutically active substances. A medically important receptor familyare G protein-coupled receptors (GPCR). They form the largest group ofmembrane-associated receptors and control the cellular response by meansof the intermediate function of G proteins. Therefore, G proteins are animportant target for drug application in the human body (Neer, E. J.,Cell, 80 (1995) 249-257; Bourne, H. R., Curr. Opin. Cell Biol., 9 (1997)134-1142; Wess, J., FASEB J, 11 (1997) 346-354).

The G_(α) sub-unit of G proteins plays a key role for the interaction ofreceptors with G proteins (Milligan, G., Mullaney, I., McKenzie, F. R.,“Specificity of interactions of receptors and effectors with GTP-bindingproteins in native membranes”, Biochem. Soc. Symp. 56, (1990) 21-34).According to the current accepted model for the activation of Gproteins, this hetero-trimeric, membrane-associated protein decomposesinto a free G_(α), a subunit bound to GTP (guanine triphospate) and afree G_(βγ)-dimer. After GTP hydrolysis, G_(a-) (GDP) again associateswith G_(βγ) (Willard F. S., Crouch M. F., Immunol. Cell Biol., 78 (2000)387-394).

Another therapeutically important class of receptors comprises channelproteins which detect extracellular signals and convert them to cellularresponses (F. M. Ashcroft: “Ion channels and disease” Academic Press,San Diego, 2000). The function of these channel proteins is to open andclose ion channels.

The existence of the endoplasmic reticulum in the cell interior is ofcrucial importance for the functionality of the signal transductionmechanism mediated by the G-protein-coupled receptors. The endoplasmicreticulum is the most important calcium store, from which calcium ions(secondary messenger compound) are released into the cytoplasm afteractivation of G-protein-coupled receptors (see e.g. Muallem, S., Wilkie,T. M., “G protein-dependent Ca²⁺ signaling complexes in polarizedcells”, Cell Calcium 26 (1999) 173-180).

Further calcium storage media of lesser importance are the cell nucleusand the mitochondria. The endoplasmic reticulum accumulates calcium ionsby means of Ca²⁺ -ATPase as an ion pump and releases calcium viacorresponding receptor ion channels, which are controlled by themessenger compounds inositol-1,4,5-triphosphate (IP3) and cyclicadenosine diphospate (cADP) (Brini, M. Carafoli, E.; “Calciumsignalling: a historical account, recent developments and futureperspectives”, Cell Mol. Life Sci., 57 (2000) 354-370). The basal Ca²⁺concentration in the endoplasmic reticulum is about 500 μM and drops toabout 100 μM when calcium ions stream into the cytoplasm (Yu, R.,Hinkle, P. M., “Rapid turnover of calcium in the endoplasmic reticulumduring signaling: studies with cameleon calcium indicators”, J. Biol.Chem., 274 (2000) 23648-23654).

An increase or decrease in the calcium concentration in a cell or avesicle can be detected, for example, using an ion-selective indicatordye.

In spite of the great importance of these proteins, there is a lack ofefficient screening assays to gain an understanding of the receptorfunctions and signal transduction processes at the molecular level, onthe one hand, and to find and develop new therapeutic compounds(“drugs”) on the other. Traditional methods of investigation are basedon assays (1) with whole cells, (2) with solubilized and purifiedreceptors, or (3) with receptors reconstituted in artificial lipidmembranes (Fernandes, P. A., Curr. Opin. Biotechnol., 9 (1998) 624-631;Zysk, J. R., Baumbach, W. R., Comb. Chem. Hight Throughput Screen., 1(1998) 171-183).

(1) In-vivo screening methods utilize adequate living biological cellswhich produce and release natural or heterologous receptor proteins atthe cell surface. In an assay, defined concentrations of test compoundsare added to the aqueous phase in the environment of the living cells,in order to investigate whether these compounds associate with receptorproteins in the cell membrane (plasma membrane) in a specific way and,for example, stimulate or inhibit a cellular response, Typical cellularresponses are changes in the intracellular ion composition (e.g. of theNa⁺ or K⁺ concentrations or of the pH), the triggering of secondarysignal cascades (e.g. the release of cAMP (cyclic adenosinemonophosphate) or of Ca²⁺) or changes in the activation of intracellularenzymes (e.g. of kinases, phosphatases, etc.). In these responses, someor all of the named processes may also occur simultaneously and/orcoupled with each other. The nature, strength and time dependence of theintracellular responses provide important information both on theinteraction between test compound and plasma membrane receptor and onthe subsequent signal transduction process. In general, the binding of atest compound to the cell surface is measured using a label associateddirectly or indirectly with this compound, or in a competitive assayusing a labeled competitor (Smith R. G., Sestili M. A., Clin. Chem., 26(1980) 543-550; Zuck P., Lao Z., Skwish S., Glickman J. F., Yang K.,Burbaum J., Inglese J., Proc. Natl. Acad. Sci. USA, 96 (1999)11122-11127). In this case, for example, it is possible to investigatewhether the binding of the test compound leads to receptor activation.

In general, an inhibition of activation is investigated in the presenceof a known agonist, by studying the effect of the test compound on theactivation of this agonist. Such standard methods are described, forexample, in: Fernandes P. B., Curr. Opin. Chem. Biol., 2 (1998) 597-603und in Gonzalez J. E., Negulescu P. A., Curr. Opin. Biotechnol., 9(1998) 624-631.

Analytical methods based on the use of living cells have a number ofinherent disadvantages: (I.) The laboratory infrastructure necessary fora continuous cultivation of cells is relatively complex. (II.) As aresult of changes in cell physiology, living biological cells areconstantly changing their properties. These changes comprise differencesin the status of the cellular growth cycle, in differentiation, and inthe strength of protein expression, making it difficult to establishreproducible, equivalent experimental conditions in parallel orrepetitive assays. (III.) A further disadvantage is, for example, thatthe possibilities for miniaturizing assays based on whole cells arelimited by the required volumes of nutrients (food) to be supplied.

In pharmacological drug screening, relatively time-consuming ligandbinding tests and receptor functionality tests are still generallyperformed separately (Hodgson, J. Bio/Technology 9 (1992) 973). On theother hand, membrane proteins, such as the G-protein-coupled receptorsand channel-forming receptors, are among the most important targetproteins for active drugs (Knowles J, “Medicines for the new milleniumhunting down diseases”, Odyssey Vol. 3 (1997)). In this context,classical patch-clamp methods are still applied as functional receptortest methods. The advantage of these electrophysiological methods isthat the function of the corresponding receptors associated withchannel-forming proteins can be measured directly. The method is highlyspecific and extremely sensitive—in principal, it is possible to measurethe channel activity of individual receptors. In this case, glassmicropipets with an opening diameter of typically 0.1-10 μm arepositioned on the surface of a biological cell. The membrane surfacethat is covered by the micropipet is called a “patch”. If the contactbetween the glass electrode and the cell membrane surface issufficiently electrically isolated, the ion current across the membranepatch can be measured electrically by means of microelectrodes which arepositioned both in the glass pipet and in the medium on the other sideof the membrane (Hamill 0. P., Marty A. et al., “Improved patch-clamptechniques for high-resolution current recording from cells andcell-free membrane patches”, Pflügers Arch., 391 (1981) 85-100).

In the context of drug screening, however, the traditional patch-clamptechnique also has crucial disadvantages. Patch-clamp measurements arevery time-consuming, require specially trained personnel with longexperience in this field and are practically not applicable for HTS(“High Throughput Screening”).

Recently, automatic methods have been developed which allow arrays ofglass microelectrodes to be positioned on suitably arranged cells or,conversely, cells or membrane fragments to be automatically (inelectrical fields or by means of suitable flow devices) positioned at(sub-)micrometer-sized apertures in solid carriers, such as glassplates, silicon wafers, the surface of which may optionally bechemically modified (Vogel H., Schmidt C., “Positionierung undelektrophysiologische Charakterisierung einzelner Zellen undrekonstituierter Membransysteme auf mikrostrukturierten Trägern”,PCT/11398/01150, WO 99/31503; Schmidt C., Mayer M., Vogel H., “Achip-based biosensor for functional analysis of single ion channels”,Angew. Chemie Int. Ed., 39 (2000) 3137-3140; Klemic K. G., Buck E. etal., “Quartz microchip partitions for improved planar lipid bilayerrecording of single channel currents”, Biophys. J 26-6A; Klemic K. G.,et al., “Design of a microfabricated quartz electrode for ultra-lownoise patch clamp recording”, Biophys. J A399).

In summary, however, it has to be concluded that the use of “native”membrane vesicles, i.e. of vesicles generated from a living cell, isdescribed for none of these methods.

(2) Assays with receptor proteins solubilized in detergents areelaborate and generally require purification from a natural source(organism) or application of a recombinant expression system. Ingeneral, the receptor activity is only partially retained in the courseof this complex procedure. In addition to receptor purification, bindingassays using fluorescent or radioactive marker molecules (“labels”) haveto be developed. By means of such binding assays, the nature of theinteraction between an agonist or antagonist and a receptor can beinvestigated, but not the stimulation or inhibition of the signaltransduction cascade, which is a central part of plasma membranereceptor function.

(3) Other traditional methods are based on receptors reconstituted inlipid vesicles. This requires first the isolation and purification ofthe receptor under consideration in suitable detergents and then theinsertion of the receptor, solubilized in a detergent, into synthetic or“native” lipid bilayer vesicles. The time-consuming purificationrequires either receptor-rich natural cells or recombinantover-expression in cells. Reconstitution protocols have to be adaptedindividually for the actual receptor under consideration. Changes in thecomposition of the lipid phase or of the aqueous phase can have a markedimpact on the efficiency of receptor insertion. The orientation of thereceptor cannot be controlled during reconstitution in the vesiclemembrane.

Both the binding of test compounds to the receptor and the activation ofion currents can be investigated using receptors reconstituted invesicles. However, these vesicles do not contain the components for thecomplete signal transduction cascade, such as G-proteins, proteinkinases, phosphatases, phosphoinosites or phospholipases, which can onlyhardly be reconstituted in vesicles together with receptors.

Therefore, there is a need for a bioanalytical reagent wherein receptorsare provided in such a form and in such a biocompatible environment thatthe complete mechanism of signal transduction associated with thereceptor under consideration is available for a bioanalyticalinvestigation method and that changes in the receptor response and/or inthe other components involved in the mechanism of signal transduction,can be tested in the presence of different biological or biochemical orsynthetic components supplied in a sample or resulting from otherchanges of other external parameters influencing the transductionmechanism. It is of major importance here to avoid the mentioneddisadvantages of assays based on whole living cells, i.e. the highvariation of the test results due to the continuous change in livingorganisms and the frequent difficulties of assigning cause-and-effectrelationships. This is due to the complex nature of whole cells whichcontain many additional biochemical components that might not bedirectly involved in the mechanism of signal transduction by a receptor,but which can also be effected in their function during a testprocedure, which may lead to further changes in the observed testresults.

According to the invention, the need for such a reagent is met byproviding a bioanalytical reagent with at least one vesicle generatedfrom a living cell comprising at least one receptor, wherein a mechanismof signal transduction triggered by said receptor in the cell used forvesicle generation is retained in said vesicle as a component of thebioanalytical reagent.

An important characteristic of many preferred embodiments of the reagentaccording to the invention is that a vesicle as a component of saidreagent comprises besides said receptor further cell products and/orcell proteins which are involved in said mechanism of signaltransduction besides said receptor, for example upon an increase ordecrease in the concentration of secondary messenger compounds. Thevesicle preferably comprises in additional further molecules suitable asindicators, which generate a signal or a corresponding signal change asa consequence of the change in concentration of these secondarymessenger compounds, which can be monitored in a bioanalytical detectionmethod.

In patent application WO 97/45534, a method is described for thecultivation of mammalian cells in the presence of immobilized vesicleswhich have been generated from a certain type of mammalian cell. In amanner similar to that described in this patent application,cytochalasin B, amongst other compounds, is used also according to thepresent invention for the generation of vesicles from living mammaliancells. However, in WO 97/45534 no hints are given to indicate the use ofsuch vesicles in bioanalytical detection methods; i.e. especially notfor the investigation of ligand-receptor interactions. The transductionmechanism of receptors that might optionally be associated with thevesicles and the influence of the method of cultivation thereon are notwithin the scope of applications described in WO 97/45534, and areconsequently not discussed in any manner therein.

The generation of vesicles from living cells upon application of thefungal toxins cytochalasin B or D is known from the literature (see forexample: Henson J. H., “Relationships between the actin cytoskeleton andcell volume regulation”, Microsc. Res. Tech., 47 (1999) 155-162; BrownS. S., Spudich J. A., “Mechanism of action of cytochalasin: evidencethat it binds to actin filament ends”, J. Cell. Biol., 88 (1981)487-491; Atlas S. J., Lin S., “Dihydrocytochalasin B. Biological effectsand binding to 3T3 cells”, J. Cell. Biol., 76 (1978) 360-370).

This method for the preparation of so-called “native” vesicles can beapplied both to adherent cells and to cells growing in suspensions.These fungal toxins act on the actin cytoskeleton. The cytoskeleton is adynamic network responsible for various essential biological functionsin the cell, such as cell division, regulation of cell volume, change ofcell form and cell movement. It has been shown that cytochalasin B and Dbind to the polymerization end of actin filaments and prevent theirextension by inhibiting the attachment of further globular actinmonomers.

After application of cytochalasin, the cells adapt a round form.Microfilaments contract and condense to local aggregates in the cellcortex. The otherwise continuous actin cytoskeleton becomes fragmentary.Supported by the cytoplasmic pressure, this effect leads to an expansionof the endoplasm in these regions. As a result, budding of the cellmembrane occurs at these locations. These buds are either bullous orpedunculate in form. In some cases without further external influences,the buds are pinched off as vesicles or can be detached from the cellsurface through the application of shear forces.

In a recent publication (Kask P., Palo K., Fay N., Brand L., Mets U.,Ullmann D., Jungmann J., Pschorr J., Gall K., “Two-dimensionalfluorescence intensity distribution analysis: theory and applications”,Biophys. J, 78 (2000) 1703-1713) the binding of fluorescent ligands to ahigh-affinity, human somatostatin receptor (SSTR-2) is demonstrated byfluorescence measurements using vesicles generated from cells(Schoeffter P., Perez J., Langenegger D., Schupbach E., Bobirnac I.,Lubbert H., Bruns C., Hoyer D., “Characterization and distribution ofsomatostatin SS-1 and SRIF-1 binding sites in rat brain: identity withSSTR-2 receptors”, Eur. J. Pharmacol., 289 (1995) 163-173). For thisstudy, membrane vesicles were generated from living cells using a glasshomogenizer. In this procedure, the cells are broken open, and thesignal transduction cascade, i.e. in particular further cell proteinsessential for signal transduction, are most likely destroyed. In theprocess, cytoplasmic vesicles are also generated, besides plasmamembrane-based vesicles. A later separation of these vesicles ofdifferent origin is not possible.—In the special case of thesomatostatin receptor the separation of the vesicle types may not beessential for the outcome of an experiment, because somatostatinreceptors are transported to the vesicle interior when they bind toagonists, and they can be localized there upon application of antibodies(Rocheville M., Lange D. C., Kumar U., Sasi R., Patel R. C., Patel Y.C., “Subtypes of the somatostatin receptor assemble as functional homo-and heterodimers”, J. Biol. Chem,. 275 (2000) 7862-7869). On the otherhand, an enrichment of the fraction of vesicles from the plasma membraneis important for applications when using receptors of only very lownatural abundance.

Contrary to the above methods, the controlled method for vesiclegeneration, according to the present invention, preserves the componentsof the signal transduction cascade, i.e. in particular, besides areceptor, the cell proteins involved in the signal transduction andtheir function also remain intact.

Said process of cell homogenization can be effected by addition ofprotease inhibitors. In general, the protease inhibitors block theenzymatic digestion of the membrane receptors by released cellproteases. The method for the production of the bioanalytical reagentaccording to the invention, upon generating vesicles using cytochalasinB, avoids the external addition of protease inhibitors. This can beadvantageous, because additional components (in this case most oftenmixtures of protease inhibitors) in a test solution may affect thereceptor-ligand interaction.

The present invention enables the adaptation of the traditional assaysand detection methods for investigation of receptor-ligand interactionsand of the subsequent signal transduction to a miniaturized format.According to the invention, the generated vesicles are used ascompartments for receptors, wherein these compartments may additionallycomprise further essential cell proteins, which may also be produced ina recombinant manner from the same cell as the vesicles to be generated.

These vesicles, as compartments derived from but independent of cells,may be frozen for storage, so that a constant quality of thesequentially used reagent aliquots of such a production lot can beassured for a lengthy period of time.

For example, the vesicles comprise either cytosolic products generatedby a recombinant method (e.g. GFP, “green fluorescent protein”) or are,for example, applied as carriers for over-expressed receptors (e.g.serotonin receptor type 3, 5HT_(3A)). According to the invention, thevesicles can also serve as compartments for other recombinant products,if they are soluble in the cytosol or comprise cell surface proteins.

To meet the demand for ever more information on intracellular andextracellular biological interactions at the molecular level, there is aneed for fast, parallel and highly sensitive analytical methods withminimum sample consumption.

For the determination of multiple analytes, the methods currently usedin particular are those wherein the detection of different analytes isperformed in discrete sample compartments or wells of so-calledmicrotiter plates. The plates most widely used in these methods arethose with an arrangement of 8×12 wells on a footprint of typically 8cm×12 cm (see, for example, Corning Costar catalogue no. 3370, 1999),wherein a volume of several hundred microliters is required for fillinga single well. For many applications, however, it would be desirable toachieve a marked reduction in the sample volume, not only to reduce therequired amount of reagents and samples, which in some cases may beavailable in only small quantities, but also to reduce the length ofdiffusion paths and thus of the assay performance times in the case ofassays in which biological or biochemical or synthetic recognitionelements for the recognition of one or more analytes in a sample areimmobilized on the wall of a sample compartment.

To reduce sample volumes and increase sample throughput, especially forscreening methods, plates with an increased density (16×24 wells=384wells and 32×48 wells 1536 wells) have been developed andcommercialized, while retaining the footprint of the standard microtiterplates. This approach allows laboratory robots which are adapted to theestablished industrial standard to be largely retained (apart from thehigher density of address points for reagent supply to the plates).Another approach was to abandon the classical plate footprint and todesign the size of the wells exclusively for the sample volumesnecessary for a certain application. Such arrangements have become knownas “nanotiter plates”, the capacity of the individual sample compartmentin some cases being no more than a few nanoliters. This technicalsolution, however, means dispensing with the currently widespreadlaboratory robots which are adapted to the classical microtiter platestandard and developing a new laboratory infrastructure optimized forthe miniaturized formats. The associated additional expense is probablyone of the main reasons for the fact that these miniaturized format haveso far not become established on the market.

As part of such methods for the detection of analytes in one or moresamples, optical methods, for example based on the determination ofchanges in absorption or luminescence, were increasingly developed inthe past, because these methods can be performed as contactlessprocedures without any major repercussions on the sample. The term“luminescence” is used in this application to denote the spontaneousemission of photons in the ultraviolet to infrared spectrum afteroptical or nonoptical excitation, such as electrical, chemical,biochemical, or thermal excitation. For example, chemiluminescence,bioluminescence, electroluminescence and especially fluorescence andphosphorescence are included in the term “luminescence”.

The classical measurement methods, such as absorption or fluorescencemeasurements, are generally based on the direct illumination of a samplevolume in a sample compartment or a measurement field on the inner wallof a sample compartment of a liquid sample. The disadvantage of thesearrangements is that, besides the excitation volume or the excitationarea wherein a signal for the detection of an analyte is to begenerated, a substantial part of the environment is generally exposed toexcitation light, which can lead to the disadvantageous generation ofinterfering background signals.

With increasing miniaturization of sample compartments, the proportionof interfering environmental light, especially as a result ofreflections or luminescence from the wall surfaces of thesecompartments, is increased further, because the relative amount of thesurface contributions to the total signal increases as the observationvolume is reduced. At the same time, the achievable sample signaldecreases in proportion to the sample volume.

In the past, essentially two approaches have been followed to improvethe ratio between the measurement signal from optionally no more than afew analyte molecules to be detected in a sample and the interferingbackground signal. One of the two approaches was to restrict theobservation volume to these few molecules and the other to restrict thedetection to that surface on which a biological or biochemicalinteraction occurs.

The first of the two approaches mentioned is based on the application ofconfocal microscopy. One example that should be mentioned isfluorescence correlation spectroscopy (FCS), developed by Eigen andRiegler, which allowed the detection of individual molecules. Using thistechnique, for example, signaling proteins in individual cells have beeninvestigated (Cluzel P., Surette M., Leibler S., “An ultrasensitivebacterial motor revealed by monitoring signaling proteins in singlecell”, Science, 287 (2000) 1652-1655). Since such studies entail thedetermination of individual processes in discrete cells or molecules,however, numerous individual measurements are necessary for aquantitative analytical conclusion to be drawn with statisticalrelevance, which overall results in a long time exposure despite thehigh sensitivity of these methods for detecting individual molecules.

This disadvantage can be avoided using the second approach, through aspatially selective analyte detection on a macroscopic interactionsurface.

Following this approach, numerous measurement arrangements have beendeveloped, wherein the detection of an analyte is based on itsinteraction with the evanescent field that is associated with lightguidance in an optical waveguide, wherein biochemical or biological orsynthetic recognition elements for specific recognition and binding ofanalyte molecules are immobilized on the surface of the waveguide. Whena light wave is coupled into an optical waveguide surrounded byoptically rarer media, i.e. media of lower refractive index, the lightwave is guided by total reflection at the interfaces of the waveguidinglayer. In this arrangement, a fraction of the electromagnetic energypenetrates the media of lower refractive index. This portion is termedthe evanescent (=decaying) field. The strength of the evanescent fielddepends to a very great extent on the thickness of the waveguiding layeritself and on the ratio of the refractive indices of the waveguidinglayer and of the media surrounding it. In the case of thin waveguides,i.e. layer thicknesses that are the same as or smaller than thewavelength of the light to be guided, discrete modes of the guided lightcan be distinguished. As an advantage of such methods, the interactionwith the analyte is limited to the penetration depth of the evanescentfield into the adjacent medium, being of the order of some hundrednanometers, and interfering signals from the depth of the (bulk) mediumcan be largely avoided. The first proposed measurement arrangements ofthis type were based on highly multimodal, self-supporting single-layerwaveguides, such as fibers or plates of transparent plastics or glass,with thicknesses from some hundred micrometers up to severalmillimeters.

For improved sensitivity and at the same time easier manufacturing inmass production, planar thin-film waveguides were used in the years thatfollowed. In the simplest case, a planar thin-film waveguide consists ofa three-layer system: support material (substrate), waveguiding layer,superstrate (i.e. the sample to be analyzed), wherein the waveguidinglayer has the highest refractive index. Additional intermediate layerscan further improve the action of the planar waveguide.

Several methods for the incoupling of excitation light into a planarwaveguide are known. The earliest methods used were based on buttcoupling or prism coupling, wherein generally a liquid is introducedbetween the prism and the waveguide in order to reduce reflectionsresulting from air gaps. These two methods are particularly suitablewith respect to waveguides of relatively large layer thickness, i.e.especially self-supporting waveguides, and with respect to waveguideswith a refractive index significantly below 2. For incoupling ofexcitation light into very thin waveguiding layers of high refractiveindex, however, the use of coupling gratings is a significantly moreelegant method.

Various methods of analyte determination in the evanescent field oflightwaves guided in optical film waveguides can be distinguished. Basedon the measurement principle applied, for example, a distinction can bedrawn between fluorescence, or more general luminescence methods on theone hand and refractive methods on the other. In this context, methodsfor the generation of surface plasmon renonance in a thin metal layer ona dielectric layer of lower refractive index can be included in thegroup of refractive methods, provided the resonance angle of thelaunched excitation light for generation of the surface plasmonresonance is taken as the quantity to be measured. Surface plasmonresonance can also be used for the amplification of a luminescence orthe improvement of the signal-to-background ratios in a luminescencemeasurement. The conditions for generation of a surface plasmonresonance and the combination with luminescence measurements, as well aswith waveguiding structures, are described in the literature, forexample in U.S. Pat. No. 5,478,755, No. 5,841,143, No. 5,006,716, andNo. 4,649,280.

In the case of the refractive measurement methods, the change in theeffective refractive index resulting from molecular adsorption to ordesorption from the waveguide is used for analyte detection. This changein the effective refractive index is determined, in the case of gratingcoupler sensors, from changes in the coupling angle for the in- orout-coupling of light into or out of the grating coupler sensor and, inthe case of interferometric sensors, from changes in the phasedifference between measurement light guided in a sensing branch and areferencing branch of the interferometer.

The aforesaid refractive methods have the advantage that they can beapplied without using additional marker molecules, so-called molecularlabels. The disadvantage of these label-free methods, however, is that,in view of the low selectivity of the measurement principle, thedetection limits achievable with these methods are confined to pico- tonanomolar concentration ranges, depending on the molecular weight of theanalyte, which is not sufficient for many applications of modern traceanalysis, for example for diagnostic applications.

For achieving lower detection limits, luminescence-based methods appearmore suitable in view of the higher selectivity of signal generation. Inthis arrangement, luminescence excitation is confined to the penetrationdepth of the evanescent field into the medium of lower refractive index,i.e to the immediate proximity of the waveguiding area, with apenetration depth of the order of some hundred nanometers into themedium. This principle is called evanescent luminescence excitation.

By means of highly refractive thin-film waveguides, based on awaveguiding film measuring only a few hundred nanometers in thickness ona transparent support material, it has been possible to increasesensitivity considerably in recent years. In WO 95/33197, for example, amethod is described, wherein the excitation light is coupled into thewaveguiding film via a relief grating as diffractive optical element.The isotropically emitted luminescence from substances capable ofluminescence, which are located within the penetration depth of theevanescent field, is measured by suitable measurement devices, such asphotodiodes, photomultipliers or CCD cameras. The portion ofevanescently excited radiation that has back-coupled into the waveguidecan also be out-coupled via a diffractive optical element, such as agrating, and be measured. This method is described, for example, in WO95/33198. The in-coupling and out-coupling grating in this method mayalso be identical, because each in-coupling grating can be used as anout-coupling grating under the same conditions as for in-coupling, inview of the reversibility of the light path.

For the simultaneous or sequential performance of exclusivelyluminescence-based, multiple measurements with essentially monomodal,planar inorganic waveguides, for example in the specification WO96/35940, arrangements (arrays) have been proposed wherein at least twodiscrete waveguiding areas are provided on one sensor platform, suchthat the excitation light guided in one waveguiding area is separatedfrom other waveguiding areas. By means of such an arrangement it ispossible, in particular, to determine different analytes simultaneouslyin an applied sample, using different recognition elements immobilizedin discrete measurement areas (d).

According to the present invention, spatially separated measurementareas (d) should be defined by the area that is occupied by biologicalor biochemical or synthetic recognition elements immobilized thereon forrecognition of an analyte in a liquid sample. These areas may have anygeometry, for example the form of dots, circles, rectangles, triangles,ellipses or lines.

For the investigation of receptor-ligand interactions, especially thefunctionality of a transduction mechanism controlled by a receptor,there is a need for a solid carrier, in particular for a sensor platformwith high detection sensitivity, designed in such a way that themechanism of signal transduction, is not impaired, in particular by theimmobilization of the receptor on a solid surface. Various embodimentsof solid carriers and/or sensor platforms are provided within the scopeof this invention.

A first subject of the invention is a bioanalytical reagent with atleast one vesicle, generated from a living cell, comprising at least onereceptor, characterized in that a mechanism of signal transductiontriggered by said receptor in the cell used for vesicle generation ispreserved in said vesicle as a component of the bioanalytical reagent.

The receptor associated with said vesicle may be located both inside thevesicle and on the vesicle membrane. It is preferred if the receptor isintegrated into the vesicle membrane.

Said mechanism of signal transduction may be triggered in this case bythe effect both of external signals or of signals inside the vesicle orof signal-generating biological or biochemical or synthetic componentspossibly added externally. “External or vesicle-internal signals” arehere understood, for example, to be changes in macroscopic properties,such as changes in ion concentrations in the medium or in the vesicle.By contrast, “signal-generating biological or biochemical or syntheticcomponents” are understood, for example, to be ligands bindingspecifically to a receptor.

An important characteristics of numerous preferred embodiments of thereagent according to the invention is that a vesicle as a component ofsaid reagent comprises further cell products and/or cell proteins,besides said one or more receptors, which are involved in said mechanismof signal transduction, besides said one or more receptors, for exampleupon an increase or a decrease of secondary messenger compounds withinthe vesicle.

Said one or more vesicles as a component of the bioanalytical reagentaccording to the invention may be generated from a eukaryotic cell orfrom a cell of native tissue.

It is preferred that the interior of said one or more vesicles is freefrom cell nucleus material, (chromosomal DNA), so that replicationprocesses do not occur within said one or more vesicles. This is animportant aspect for applications with “native” vesicles, which are freeof heterologous DNA (for example upon the insertion of vesicles as acarrier into another target organism).

For many applications of the bioanalytical reagent according to theinvention, it is preferred that said one or more vesicles have adiameter of 50 nm-5000 nm. A diameter of 100 m-2000 nm is especiallypreferred.

A characteristic of many embodiments of the bioanalytical reagentaccording to the invention is that said one or more receptors arepresent in natural form in said one or more vesicles as a component ofthe bioanalytical reagent.

For other applications it is preferred that said one or more receptorsare present in a modified form in said one or more vesicles as acomponent of the bioanalytical reagent. For example, a receptor may bepresent as a fusion protein, for example by fusion with a fluorescentprotein such as GFP (green fluorescent protein, Tsien R. Y., “The greenfluorescent protein”, Annu. Rev. Biochem. 67 (1998) 509-544) or BFP(blue fluorescent protein) or RFP (red fluorescent protein).

It is characteristic of many embodiments that said one or more receptorsare present in recombinant form in said one or more vesicles as acomponent of the bioanalytical reagent.

An important characteristic of the bioanalytical reagent according tothe invention is that a binding capability of said one or more receptorsto a specific ligand is preserved, this binding capacity being presentin said vesicle-generating cell and the receptor being associated withthe vesicle as a component of the bioanalytical reagent.

The one or more receptors may be selected from the group ofsignal-transducing receptors that is formed by plasma membranereceptors, such as ion channel receptors, G-protein-coupled receptors(GPCR), orphan receptors, enzyme-coupled receptors, such as receptorswith an intrinsic tyrosine kinase activity, receptors with an intrinsicserine/threonine kinase activity, furtheron by receptors for growthfactors (peptide hormone receptors), receptors for chemotacticsubstances, such as the class of chemokine receptors, and byintracellular hormone receptors, such as steroid hormone receptors.

Characteristic of some embodiments of the bioanalytical reagentaccording to the invention is that said one or more receptors are incontact with the outer vesicle membrane. It is then preferred that, withrespect to the surface of the outer vesicle membrane, the areal densityof receptors that are in contact with the outer vesicle membrane is ofsimilar order of magnitude or greater than the corresponding density ofthese receptors in the vesicle-generating living cell.

Characteristic of other embodiments of the bioanalytical reagentaccording to the invention, by contrast, is that said one or morereceptors are located in the interior of the vesicle. In this case, itis preferred that, with respect to the vesicle volume, the volumedensity of receptors located in the interior of a vesicle is of similarorder of magnitude or greater than the density of the receptors in thevesicle-generating living cell.

It is of course particularly advantageous if, in the sense of anenrichment of receptors and/or of their ligand binding sites, theprocess of production of the vesicle from a living cell allows saidareal density of the receptors in the vesicle membrane in the case ofreceptors being in contact with the outer vesicle membrane, or theirvolume density inside the vesicle in the case of ligand binding siteslocated in the vesicle interior, to be increased with respect to thecorresponding densities in the original cell.

It is preferred that said one or more vesicles comprise, besides saidone or more receptors, further biological compounds (components) fromthe group that is formed e.g. by G proteins and G-protein regulators(e.g. rasGAP), enzymes such as adenylate cyclases, phospholipases whichform intracellular secondary messenger compounds (e.g. cAMP (cyclicadenosine monophosphate), cGMP (cyclic guanosine monophosphate), diacylglycerol (DAG) or inositol triphosphate (IP3)), enzymes such as serine,threonine and tyrosine kinases, and tyrosine phosphatases that activateor inhibit proteins by phosphorylation or de-phosphorylation.

The bioanalytical reagent according to the invention is also suitablefor application in a living organism, for example, to performbioanalytical studies in the organism at a specific, pre-determinedsite, for example by means of indicator compounds incorporated in thevesicle. For example it is advantageous for such applications ifbiological, biochemical or synthetic compounds, such as cell surfaceproteins or cell surface sugars, are associated with the outer membraneof the one or more vesicles, these compounds being used for thetransport of said vesicle to pre-determined destinations, such as cellsand/or organs and/or pre-determined tissue in a living organism, and/orfor the binding to a biological or biochemical or synthetic recognitionelement which specifically recognizes and binds said biological orbiochemical or synthetic recognition element.

In order to reduce nonspecific binding of a vesicle, as a component of abioanalytical reagent according to the invention, to a surface broughtinto contact with the vesicle, it may be advantageous if lipidscomprising, for example, hydrophilic polymers (such as polyethyleneglycols) are additionally integrated into the vesicle membrane aftergeneration of said vesicle from a living cell. Vesicles withsurface-associated polymers are described, for example, in theinternational application PCT/EP 00/04491.

Said mechanism of signal transduction, which is preserved in thebioanalytical reagent according to the invention, may comprise amechanism from among the group of mechanisms that is formed e.g. fromion conducting, G-protein coupling, activation or inhibition ofintra-vesicular ion channels, intra-vesicular release of calcium,protein activation or inhibition by enzymatic phosphorylation orde-phosphorylation (kinase cascades; phosphatases), and release orenzymatic formation of secondary messenger compounds, such as cAMP, cGMPor diacyl glycerol (DAG), inositol triphosphate (IP3).

Said mechanism of signal transduction may comprise a (secondary)functional response of the one or more vesicle-associated receptorsafter a primary specific interaction of said receptor with one or morenatural and/or synthetic ligands contained in a sample that is broughtinto contact with said vesicle.

The mechanism of signal transduction may also comprise the activation ofan ion channel of a receptor associated with a vesicle, as a componentof said bioanalytical reagent.

It is also possible that said mechanism of signal transduction comprisesthe binding of a G-protein to a receptor associated with a vesicle, as acomponent of said bioanalytical reagent.

Said mechanism of signal transduction may also comprise the internalrelease of ions, such as Ca²⁺, or of other messenger compounds, such ascAMP or cGMP.

The mechanism of signal transduction may also comprise the enzymaticdecomposition of a substrate by a vesicle-associated enzyme to form aproduct. In this case, said vesicle may be located in the vesicleinterior or may be associated with the vesicle membrane.

Characteristic of a specific embodiment of the bioanalytical reagentaccording to the invention is that a (secondary) functional response aspart of said mechanism of signal transduction occurs after interactionbetween one or more natural and/or synthetic ligands or co-factorscontained in a sample brought into contact with said vesicle on the onehand and naturally or recombinantly generated proteins associated withsaid vesicle on the other.

It is preferred that said one or more vesicles additionally comprisecomponents for generation of an experimentally detectable signal.

These additional components for generation of an experimentallydetectable signal may be associated with further biological compounds(components) which are associated in turn with the one or more vesiclesas a component of said bioanalytical reagent.

In this case, said further biological compounds (components) mayoriginate from the group that is formed e.g. by G proteins and G-proteinregulators (e.g. rasGAP), enzymes such as adenylate cyclases,phospholipases which form intracellular secondary messenger compounds(e.g. cAMP (cyclic adenosine monophosphate), cGMP (cyclic guanosinemonophosphate), diacyl glycerol (DAG) or inositol triphosphate (IP3)),enzymes such as serine, threonine and tyrosine kinases, and tyrosinephosphatases that activate or inhibit proteins by phosphorylation orde-phosphorylation.

The said additional components for generation of an experimentallydetectable signal may also be associated with a receptor or may be partsof fusion proteins associated with the vesicle.

Said additional components for generation of an experimentallydetectable signal may be selected from the group of components formed byabsorptive indicators and luminescent indicators, luminescence labels,luminescent nanoparticles, absorptive indicator proteins and luminescentindicator proteins, such as BFP (“blue fluorescent protein”), GFP(“green fluorescent protein”) or RFP (“red fluorescent protein”),artificial luminescent (i.e. in particular fluorescent) amino acids,radioactive labels, spin labels, such as NMR labels or ESR labels, ionindicators, especially pH and calcium indicators, or potential-dependentindicators, such as potential-dependent luminescence labels, or redoxcomplexes.

Characteristic of a preferred embodiment of the bioanalytical reagentaccording to the invention is that said additional components forgeneration of an experimentally detectable signal are generated from thesame cell from which the vesicle was generated.

Characteristic of another possible embodiment is that said additionalcomponents for generation of an experimentally detectable signal areinserted into the cell from which the vesicle is generated beforeproduction of the vesicle.

However, said additional components for generation of an experimentallydetectable signal may also be inserted into the vesicle after itsformation.

A very important and advantageous characteristic for the application ofthe bioanalytical reagent according to the invention in practice is thatthe functionality of a receptor associated with a vesicle as a componentof said bioanalytical reagent is preserved upon storage underdeep-frozen conditions for at least one week, preferably for at leastone month, especially preferably for at least one year. Preciseconditions for the deep-freezing of vesicles as part of a bioanalyticalreagent according to the invention are described in Example 3. In thiscase, the “preservation of the functionality” of said receptor isintended to mean that a mechanism of signal transduction to be triggeredby said receptor in the vesicle is also when the vesicle is thawed outagain intact after storage of the vesicle under the conditionsdescribed.

Exposed to different conditions, for example under sterile conditions incooled buffer solution, i.e. at a temperature below ambient temperature,e.g. at 4° C., a bioanalytical reagent according to the invention ischaracterized by a shelf life of at least one week.

A further subject of the present invention is a method for production ofa bioanalytical reagent with a vesicle generated from a living cellaccording to any of the embodiments mentioned above, wherein saidvesicle was produced from a living cell comprising at least onereceptor, and wherein a mechanism of signal transduction triggered bysaid receptor in said living cell is preserved in said vesicle as acomponent of the bioanalytical reagent.

It is preferred if the constriction and pinching off of said vesiclefrom said living cell is effected after application of cytochalasin Band/or cytochalasin D.

It is also preferred if the method according to the invention isperformed without application of protease inhibitors. In the case ofother methods wherein cell proteases are released, there is the riskthat a certain decomposition of receptor proteins may occur despite theaddition of a protease inhibitor or mixtures of protease inhibitors.Protease inhibitors also have to be removed in additional purificationsteps.

The method according to the invention may comprise the application ofshear forces and/or of centrifugation steps, for example upon exposureto a gradient of sucrose, and/or the application of chromatographicsteps, for example by separation into fractions of different sizedistributions, and/or the application of filtration steps and/or theapplication of electrophoretic methods.

By means of the method according to the invention, said one or morevesicles may be generated from a eukaryotic cell. Said one or morevesicles may also be generated from a cell of native tissue.

Characteristic of an important embodiment of the method according to theinvention is that the interior of a vesicle produced by said method isfree of cell nucleus material, so that replicative processes do notoccur.

For a preferred embodiment of the method according to the invention, itis characteristic that a vesicle produced by said method has a diameterof 50 nm-5 000 nm, especially preferably of 100-2 000 nm.

For many applications, it is preferred if a receptor in a vesicleproduced by this method, as a part of the bioanalytical reagent, isprovided in natural form.

For other applications it is preferred that a receptor in a vesicleproduced by this method, as a part of the bioanalytical reagent, isprovided in a modified form. Said receptor, may be, for example,provided as a fusion protein, e.g. by fusion of a fluorescent proteinsuch as GFP (green fluorescent protein) or BFP (blue fluorescentprotein) or RFP (red fluorescent protein). Another example is the fusionof a GPCR and a G-protein.

Often it is also advantageous if a receptor in a vesicle produced bythis method is provided in recombinant form.

An important characteristic of the method according to the inventioncomprises the preservation of a binding capability of said one or morereceptors to a specific ligand, this binding capability being present insaid vesicle-generating cell and the receptor being associated with thevesicle as a component of the bioanalytical reagent.

The one or more receptors may be selected from the group ofsignal-transducing receptors that is formed by plasma membranereceptors, such as ion channel receptors, G protein-coupled receptors(GPCR), orphan receptors, enzyme-coupled receptors, such as receptorswith an intrinsic tyrosine kinase activity, receptors with an intrinsicserine/threonine kinase activity, furtheron by receptors for growthfactors (peptide hormone receptors), receptors for chemotacticsubstances, such as the class of chemokine receptors, and byintracellular hormone receptors, such as steroid hormone receptors.

Preferred are embodiments of the method according to the inventionwherein, with respect to the surface of the outer vesicle membrane, theareal density of receptors that are in contact with the outer vesiclemembrane is of a similar order of magnitude or greater than thecorresponding density of these receptors in the vesicle-generatingliving cell.

Also preferred are embodiments of the method wherein, with respect tothe vesicle volume, the volume density of receptors located in theinterior of a vesicle is of similar order of magnitude or greater thanthe density of these receptors in the vesicle-generating living cell.

Characteristic of further embodiments of the method according to theinvention is that said one or more vesicles produced by this methodcomprise, besides said one or more receptors, further biologicalcompounds (components) from the group that is formed e.g. by G proteinsand G-protein regulators (e.g. rasGAP), enzymes such as adenylatecyclases, phospholipases which form intracellular secondary messengercompounds (e.g. cAMP (cyclic adenosine monophosphate), cGMP (cyclicguanosine monophosphate), diacyl glycerol (DAG) or inositol triphosphate(IP3)), enzymes such as serine, threonine and tyrosine kinases, andtyrosine phosphatases that activate or inhibit proteins byphosphorylation or de-phosphorylation.

Characteristic of further embodiments of the method is that biological,biochemical or synthetic compounds, such as cell surface proteins orcell surface sugars, are associated with the outer membrane of the oneor more vesicles produced by this method, these compounds being used forthe transport of said vesicle to pre-determined destinations, such ascells and/or organs and/or pre-determined tissue in a living organism,and/or for the binding to a biological or biochemical or syntheticrecognition element, which specifically recognizes and binds saidbiological or biochemical or synthetic recognition element.

To reduce nonspecific binding of a vesicle, as part of a bioanalyticalreagent according to the invention, to a surface that is to be broughtinto contact with the vesicle, it may be advantageous if, afterproduction of said vesicle from a living cell, lipids comprising forexample hydrophilic polymers (such as polyethylene glycols) areadditionally integrated into the vesicle membrane. Vesicles withsurface-associated polymers are described, for example, inPCT/EP/00/04491.

Of high importance are also embodiments of the method according to theinvention wherein said one or more vesicles produced by this methodadditionally comprise components for generation of an experimentallydetectable signal.

These additional components for generation of an experimentallydetectable signal may be comprised in the further biological compounds(components) associated with the one or more vesicles as part of saidbioanalytical reagent. In this case, said further biological compounds(components) may be comprised in the group that is formed e.g. by Gproteins and G-protein regulators (e.g. rasGAP), enzymes such asadenylate cyclases, phospholipases which form intracellular secondarymessenger compounds (e.g. cAMP (cyclic adenosine monophosphate), cGMP(cyclic guanosine monophosphate), diacyl glycerol (DAG) or inositoltriphosphate (IP3)), enzymes such as serine, threonine and tyrosinekinases, and tyrosine phosphatases that activate or inhibit proteins byphosphorylation or de-phosphorylation.

Said additional components for generation of an experimentallydetectable signal my also be associated with a receptor or may be partsof fusion proteins associated with the vesicle.

Said additional components for generation of an experimentallydetectable signal may be selected from the group of components formed byabsorptive indicators and luminescent indicators, luminescence labels,luminescent nanoparticles, absorptive indicator proteins and luminescentindicator proteins, such as BFP (“blue fluorescent protein”), GFP(“green fluorescent protein”) or RFP (“red fluorescent protein”),artificial luminescent (i.e. in particular fluorescent) amino acids,radioactive labels, spin labels, such as NMR labels or ESR labels, ionindicators, especially pH and calcium indicators, or potential-dependentindicators, such as potential-dependent luminescence labels, or redoxcomplexes.

According to the method according to the invention, said additionalcomponents for generation of an experimentally detectable signal may begenerated from the same cell from which the vesicle was produced.

The cell may also be loaded with said additional components forgeneration of an experimentally detectable signal before production ofthe vesicle.

However, said additional components for generation of an experimentallydetectable signal may also be inserted into the vesicle after itsformation.

A further subject of the invention is a bioanalytical detection methodwith a bioanalytical reagent according to any of the aforementionedembodiments, wherein said detection method is selected from the groupthat is formed, for example, by optical detection methods, such asrefractrometric methods, surface plasmon resonance, optical absorptionmeasurements (e.g. internal reflection methods using a highly refractivematerial, in combination with infrared spectroscopic measurements) orluminescence detection (e.g. fluorescence correlation spectroscopy),detection of energy or charge transfer, mass spectroscopy, electrical orelectrochemical detection methods, such as electrophysiology, patchclamp techniques, impedance measurements, electronic resonancemeasurements, such as electron spin resonance or nuclear spin resonance,gravimetric methods (e.g. electrical crystal balance measurements),radioactive methods, or by electrophoretic measurements.

Characteristic of some of the possible embodiments of the bioanalyticaldetection method according to the invention is that said method isperformed in homogeneous solution.

A special group of embodiments of the bioanalytical detection methodaccording to the invention with a bioanalytical reagent according to theinvention is related to special patch-clamp methods which can beperformed in a single-device arrangement or in a multiple-devicearrangement. Characteristic of a bioanalytical detection methodaccording to this embodiment is that said method is performed using ameasurement arrangement with at least 2 electrodes and separatecompartments suitable for receiving liquids, wherein a solid carrier(preferably as an electrically isolating separation wall), comprising atleast one aperture and separating at least 2 compartments, is locatedbetween two electrodes facing each other, the electrodes being of anygeometrical form and each extending into at least one compartment orbeing in contact with at least one compartment.

Said carrier is provided as a separation wall comprising an electricallyisolating material located between the electrodes. As mentioned above,the carrier is provided with an aperture and with a surface on whichvesicles from a bioanalytical reagent according to the invention can befixed. The carrier must not necessarily consist of a single piece, butit may e.g. comprise a holder to which the material which is actuallyrelevant for membrane binding and membrane positioning may be attachedor in which this material may be inserted, said material comprising atleast one aperture for the binding and positioning of the membranes.Additionally, the aperture may be surrounded by a circular, taperingelevation (typically with a height in the sub-micrometer range and theaperture located in the center). Thus, a micropipet-like aperture may begenerated on an otherwise essentially planar carrier.

In the presence of a potential difference over the measurementarrangement and mediated by the two or more electrodes, such a specificarrangement allows an inhomogeneous electrical field to be generatedaround the aperture, said field having an increasing value withdecreasing distance from the aperture and said field being capable ofmoving vesicles electrophoretically towards the aperture, said vesiclesbeing located close to said aperture and generated from a living cell,from a bioanalytical reagent according to the invention.

Such an arrangement also allows such vesicles to be positioned over orwithin the aperture by means of a hydrodynamic or electrokinetic flow orby other mechanical manipulation (e.g. by means of optical tweezers,force microscope or by a micro manipulator).

The fixation of the membranes may for example be based on electrostaticinteractions between e.g. a negatively charged membrane surface and apositively charged carrier surface. If the carrier surface by itself isnot provided with the desired charge, it may be modified accordingly.

It is preferred if said measurement arrangement is provided with meanson one side or on both sides of the carrier which enable a supply ofliquid and/or a storage of liquid and/or an exchange of liquid and/orthe addition of vesicles generated from a living cell, from abioanalytical reagent according to the invention, between carrier andelectrode.

It is also preferred if the one or more apertures of said measurementarrangement have such a diameter that, in the presence of a potentialdifference and mediated by the two or more electrodes, an inhomogeneouselectrical field is generated around the aperture, said field having anincreasing value with decreasing distance from the aperture and saidfield being capable of moving vesicles electrophoretically towards theaperture, said vesicles being located close to said aperture andgenerated from a living cell, from a bioanalytical reagent according tothe invention.

It is also advantageous if the one or more apertures of said measurementarrangement have such a diameter that vesicles generated from a livingcell, from a bioanalytical reagent according to the invention, can bepositioned over or within the aperture by means of a hydrodynamic orelectrokinetic flow or by other mechanical manipulation (e.g. by meansof optical tweezers, force microscope or by a micro manipulator).

Characteristic of many embodiments of this special group ofbioanalytical detection methods according to the invention is that thecarrier of said measurement arrangement is provided with an electricallycharged surface which exerts attractive force on vesicles generated froma living cell, from a bioanalytical reagent according to the invention,or is provided with an adhesion-promoting layer for binding saidvesicles on its surface.

The bioanalytical detection method according to the invention may beperformed by inserting vesicles generated from a living cell, from abioanalytical reagent according to the invention, between separationwall or carrier and electrode into a compartment filled or not filledwith buffer beforehand, and by moving said vesicles towards the apertureby means of an electrical potential difference applied to theelectrodes, and/or by positioning said vesicles on the aperture byhydrodynamic or electrokinetic flow and/or by positioning the vesicleson the aperture mechanically (e.g. by means of optical tweezers, forcemicroscope or by a micro manipulator).

In particular it is possible that vesicles generated from a living cell,from a bioanalytical reagent according to the invention, are positionedon said aperture, the vesicle membranes form an electrically closecontact with the carrier over the aperture, and a measurement of the(electrical) membrane resistance is enabled. During this procedure, thevesicles may preserve their form or may merge with the surface of thecarrier and thus form an aperture-spanning planar membrane. In thiscase, a good signal-to-noise discrimination can be achieved by means ofthe method according to the invention.

The method also allows artificial lipid vesicles with a diameter largerthan the diameter of said aperture to be added to at least onecompartment, in order to generate a planar lipid bilayer on the surfaceof the carrier and extending over the aperture, and vesicles generatedfrom a living cell, from a bioanalytical reagent according to theinvention, then to be added to said compartment, in order to fuse saidvesicles with the generated lipid membrane and to render receptors thatare associated with said vesicles generated from living cells accessiblefor electrical or optical measurements.

Moreover, the method enables membrane proteins to be inserted into avesicle generated from a living cell, after positioning said vesicle onan aperture.

It is preferred if a vesicle generated from a living cell located overan aperture or a planar membrane generated from said vesicle andspanning an aperture is accessible for optical measurements, especiallyfor fluorescence measurements, or for simultaneous optical andelectrical measurements, to which it is subjected.

Characteristic of a special variant of this method according to theinvention based on patch-clamp techniques is that a measurementarrangement or a measurement system with several apertures on onecarrier is used and that measurements on at least two apertures areperformed sequentially and/or in parallel.

In particular, numerous vesicles generated from living cells, from abioanalytical reagent according to the invention, may be arranged in anarray on a solid, electrically isolating carrier, wherein said array ofvesicles is brought into electrically isolating contact with an array ofpatch-clamp pipets in a geometrical arrangement similar to that of thevesicle array, in order to enable a simultaneous performance ofelectrical measurements independently of each other or simultaneouselectrical and optical measurements on a large number of individualvesicles.

Further embodiments of such measurement arrangements, especially with a“patch-clamp array” and analytical detection methods based on the usethereof, which are suitable for a bioanalytical detection methodaccording to the invention using a bioanalytical reagent according tothe invention, are described in WO 99/31503. The use of thesemeasurement arrangements and detection methods, in combination with abioanalytical detection method according to the invention using abioanalytical reagent according to the invention, is also a subject ofthe present invention.

Characteristic of numerous possible embodiments of a bioanalyticaldetection method according to the invention is that the one or morevesicles generated from a living cell, comprising at least one receptor,from a bioanalytical reagent according to the invention, is immobilizedon the surface of a solid support.

It is advantageous if a vesicle generated from a living cell, from abioanalytical reagent according to the invention, is also accessible inparticular for mass-spectrometric investigations after immobilization ona solid support.

Characteristic of said embodiments of a bioanalytical detection methodaccording to the invention is that a mechanism of signal transductiontriggered by said receptor in said living cell is preserved in a vesiclegenerated from the cell after immobilization of the vesicle.

A particular subject of the invention is therefore a bioanalyticaldetection method with at least one vesicle immobilized on the surface ofa solid support, the vesicle being generated from a living cell,comprising at least one receptor, from a bioanalytical reagent accordingto the invention, wherein a mechanism of signal transduction triggeredby said receptor in said living cell is preserved in a vesicle generatedfrom the cell after immobilization of the vesicle.

It is preferred if vesicles, each comprising at least one receptor, areimmobilized in discrete measurement areas (d) with one or more vesicleseach on the surface of said solid support.

It is further preferred if vesicles with at least two different kinds ofreceptor are immobilized in numerous measurement areas (d), wherein eachvesicle is preferably immobilized with the same kind of receptor withinan individual measurement area.

The one or more vesicles generated from a living cell may be immobilizedon the surface of said solid support for example by means of covalentbinding or physical adsorption (electrostatic or van der Waalsinteraction or hydrophilic or hydrophobic interaction or a combinationof these interactions).

It is preferred if an adhesion-promoting layer is deposited between thesurface of said solid support and the one or more vesicles immobilizedthereon. In this case, according to the invention, theadhesion-promoting layer is designed in such a way that a mechanism ofsignal transduction triggered by the one or more receptors in saidliving cell is preserved also after immobilization of the vesiclesgenerated from a living cell as part of a bioanalytical reagentcomprising at least one receptor, according to the invention, on saidadhesion-promoting layer.

It is preferred if the adhesion-promoting layer has a thickness ofpreferably less than 200 nm, most preferably of less than 20 nm.

The adhesion-promoting layer may comprise a chemical compound of thegroup of silanes, epoxides, functionalized, charged or polar polymersand “self-organized functionalized mono or multiple layers”.

Characteristic of a preferred embodiment is that the adhesion-promotinglayer comprises a monomolecular layer of mainly one kind of protein,such as serum albumins or streptavidin, or of modified proteins, such asbiotinylated serum albumin.

Characteristic of another preferred embodiment is that theadhesion-promoting layer comprises self-organized alkane-terminatedmonolayers of mainly one kind of chemical or biochemical molecules.

Especially preferred is an embodiment wherein the adhesion-promotinglayer is provided as a double layer (bilayer), comprising an initialself-organized alkane-terminated anchoring layer and a second layerformed by self-organization (self-assembly) of synthetic or naturallipids.

The immobilization of the one or more vesicles generated from a livingcell on the adhesion-promoting layer may be performed, for example, uponcovalent binding or upon physical adsorption (electrostatic or van derWaals interaction or hydrophilic or hydrophobic interaction or acombination of these interactions).

A special embodiment of the bioanalytical detection method according tothe invention, using a particularly specific variant of vesicleimmobilization, comprises association with the adhesion-promoting layerof biological or biochemical or synthetic recognition elements whichrecognize and bind a vesicle generated from a living cell withsurface-associated biological or biochemical or synthetic components forspecific recognition and binding, as part of the correspondingabove-described specific embodiment of a bioanalytical reagent accordingto the invention. These specific interactions for the recognition andbinding of the vesicles to their recognition elements on theadhesion-promoting layer may for example be based on interactions withbiotin/streptavidin, so-called “histidine tags” (references: Schmid E.L., Keller T. A., Dienes Z., Vogel H., “Reversible oriented surfaceimmobilization of functional proteins on oxide surfaces”, Anal Chem 69(1997) 1979-1985; Sigal G. B., Bamdad C., Barberis A., Strominger J.,Whitesides G. M., “A self-assembled monolayer for the binding and studyof histidine-tagged proteins by surface plasmon resonance”, Anal Chem 68(1996) 490-497), sugars or peptide affinity interactions, wherein anyone of the two binding partners in each case may be associated with thevesicle surface and the other anchored on the surface of saidadhesion-promoting layer.

Characteristic of another embodiment of the bioanalytical detectionmethod according to the invention is that at least one ligand for areceptor, which is bound to a vesicle generated from a living cell, froma bioanalytical reagent according to the invention, is immobilized,optionally by means of a spacer molecule, on the surface of the solidsupport.

In this case it is preferred if at least two different ligands forreceptors, which are bound to a vesicle generated from a living cell,from a bioanalytical reagent according to the invention, are immobilizedin numerous measurement areas (d), wherein preferably the same kind ofligand is immobilized within an individual measurement area.

Said ligands may be immobilized on the surface of the solid support bymeans of covalent binding or physical adsorption (e.g. electrostatic orvan der Waals interaction or hydrophilic or hydrophobic interaction or acombination of these interactions).

It is preferred if an adhesion-promoting layer is applied between thesurface of the solid support and said ligands immobilized thereon.

For the selection of the adhesion-promoting layer for immobilization ofsaid ligands, the same preferences apply as mentioned above for anadhesion-promoting layer for immobilization of vesicles generated from aliving cell, from a bioanalytical reagent according to the invention.

Characteristic of a particularly preferred embodiment of a bioanalyticaldetection method according to the invention is that regions between thelaterally separated measurement areas, with vesicles generated fromliving cells (from a bioanalytical reagent according to any of thedescribed embodiments) immobilized therein, or with ligands forreceptors that are bound to vesicles generated from living cells (from abioanalytical reagent according to any of the described embodiments)immobilized therein, and/or that regions within these measurement areas,between the compounds immobilized therein, are “passivated” in order tominimize nonspecific binding of analytes or of their detection reagents,i.e., that compounds which are “chemically neutral” towards the analyteare deposited between the laterally separated measurement areas (d)and/or within these measurement areas (d) between said immobilizedcompounds, the “chemically neutral” compounds preferably being composedof the groups that are formed by albumins, casein, detergents, such asTween 20, detergent/lipid mixtures (of synthetic and/or natural lipids),synthetic and natural lipids or also hydrophilic polymers, such aspolyethylene glycols or dextrans.

It is also possible to passivate an activated surface (activated forimmobilization of the biological, biochemical recognition elements, theactivated surface comprising e.g. poly-L-lysin or functionalized silanescomprising e.g. aldehyde or epoxy groups), for example by the additionof reducing reagents such as sodium borate (in the case of aldehyde orepoxy groups).

The material of the surface of the solid support (carrier) withimmobilized vesicles generated from living cells (from a bioanalyticalreagent according to the invention and any of the described embodiments)or with immobilized ligands for receptors that are bound to vesiclesgenerated from living cells (from a bioanalytical reagent according tothe invention and any of the described embodiments) may comprise amaterial of the group which is formed e.g. by moldable, sprayable ormillable plastics, carbon compounds, metals, such as gold, silver,copper, metal oxides or silicates, such as glass, quartz or ceramics, orsilicon or germanium or ZnSe or a mixture of these materials.

In this case, said solid carrier (support) may be provided in variousembodiments. It may be provided e.g. as a glass or microscope plate. Itmay also be a microtiter plate of the type for example that is inwidespread use for screening assays (for testing numerous compounds,e.g. using classical fluorescence methods or fluorescence correlationspectroscopy).

It is preferred if the surface of said solid support (carrier) isessentially planar.

Characteristic of a preferred group of embodiments of the bioanalyticaldetection method is said solid support (carrier) is an optical orelectronic sensor platform.

In this case, it is preferred that said solid support (carrier) istransparent at least in a region of wavelengths in the ultraviolet toinfrared spectrum and comprises preferably a material from the groupthat is formed e.g. by moldable, sprayable or millable plastics, carboncompounds, metals, metal oxides or silicates, such as glass, quartz orceramics, or silicon or germanium or ZnSe or a mixture of thesematerials.

It is preferred here if said solid support is an optical waveguide usedas a sensor platform. Specially preferred is an embodiment of thedetection method wherein said solid support is an optical thin-filmwaveguide used as a sensor platform, with an initial opticallytransparent layer (a) with refractive index n₁ on a second opticallytransparent layer (b) with refractive index n₂, wherein n₁>n₂.

For example for the simultaneous analysis of multiple samples and/or forthe determination of multiple analytes in one or more samples it isadvantageous, if the sensor platform as a solid support is divided intotwo or more discrete waveguiding regions.

The material of the second optically transparent layer (b) of the sensorplatform as a solid support may be selected from the group that isformed by silicates, such as glass or quartz, or transparent moldable,sprayable or millable, especially thermoplastic plastics, such aspolycarbonates, polyimides, polymethyl methacrylates, or polystyrenes.

It is preferred if the refractive index of the first opticallytransparent layer (a) of the sensor platform as a solid support isgreater than 1.8.

It is further preferred if the first optically transparent layer (a) ofthe sensor platform as a solid support comprises TiO₂, ZnO, Nb₂O₅,Ta₂O₅, HfO₂, or ZrO₂, preferably TiO₂ or Ta₂O₅ or Nb₂O₅.

The first optically transparent layer (a) preferably has a thickness of40 to 300 nm, most preferably of 100 to 200 nm.

Characteristic of a further embodiment of the bioanalytical detectionmethod according to the invention is that an additional opticallytransparent layer (b′) with lower refractive index than layer (a) andwith a thickness of 5 nm-10 000 nm, preferably of 10 run-1000 nm, islocated between the optically transparent layers (a) and (b) and incontact with layer (a). This intermediate layer (b′) can for exampleserve to improve the adhesion of layer (a) on layer (b) or to reduce theeffect of surface roughnesses of layer (b). However, layer (b′) can alsoserve to reduce the penetration of the evanescent field of light guidedin layer (a) into layer (b), for example in order to reduce an unwantedluminescence excitation in layer (b).

Characteristic of a preferred embodiment of the bioanalytical detectionmethod according to the invention is that the in-coupling of excitationlight into the optically transparent layer (a) to the measurement areas(d) on the sensor platform as a solid support is performed using one ormore optical in-coupling elements from the group formed by prismcouplers, evanescent couplers comprising joined optical waveguides withoverlapping evanescent fields, butt-couplers with focusing lenses,preferably cylindrical lenses, arranged in front of a front face (distalend) of the waveguiding layer, and grating couplers.

It is especially preferred in this case that the in-coupling ofexcitation light into the optically transparent layer (a) to themeasurement areas (d) is performed using one or more grating structures(c) that are formed in the optically transparent layer (a).

Subject of the invention in a more general form is a bioanalyticaldetection method according to any of the embodiments described above,wherein one or more liquid samples comprising vesicles generated fromliving cells (from a bioanalytical reagent according to the invention)with associated receptors are brought into contact with the ligands forthese receptors immobilized in one or more measurement areas, and asignal change caused by a binding of the receptors associated with saidvesicles to their immobilized ligands is measured.

It is preferred in this case if the signal transduction of receptorsassociated with vesicles generated from living cells (from abioanalytical reagent according to the invention), is measured afterbinding of these receptors to their immobilized ligands, wherein thissignal transduction may be triggered, for example, by binding of furtherligands to the receptors associated with said vesicles or by otherinducing influences.

For example, in multiple measurement areas a uniform kind of ligand maybe immobilized. If these measurement areas individually or several ofthese measurement together can be fluidically addressed, for example,within sample compartments comprising the solid carrier (support) as abase plate (see also below), screening methods based on the use of abioanalytical reagent according to the invention are facilitated thatare interesting for industrial research and development. For example,different vesicles generated from different living cells, withoptionally different associated receptors and optionally differentadditional biological compound (components) may be supplied to differentmeasurement areas with similar ligands immobilized therein, and thedifferent binding behavior to the immobilized ligands in the discretemeasurement areas may be investigated.

Characteristic of a further preferred embodiment of this bioanalyticaldetection method according to the invention is that the binding ofreceptors that are associated with vesicles generated from living cells(from a bioanalytical reagent according to the invention) to saidimmobilized ligands occurs in competition with the binding of thesereceptors associated with said vesicles to ligands in free solution.

If these measurement areas individually or several of these measurementareas together can be fluidically addressed, for example, within samplecompartments comprising the solid carrier (support) as a base plate (seealso below), it is also possible to supply to individual samplecompartments different concentrations of vesicles generated from livingcells in a bioanalytical reagent according to the invention, and toinvestigate the competition between the binding of the associatedreceptors to the immobilized ligands and to ligands located in freesolution. Another variant comprises generating different surfaceconcentrations of ligands immobilized in measurement areas, to which auniform concentration of vesicles in a bioanalytical reagent accordingto the invention is added, and then to investigate again the competitionbetween the binding of the associated receptors to the immobilizedligands and to the ligands in free solution.

It is obvious for those skilled in the art that a variety of furtherpossible embodiments results from the combination of the aforementionedvariants of bioanalytical detection methods according to the invention.

The aforementioned variants using ligands immobilized on a solid carrier(support) to which vesicles generated from living cell, from abioanalytical reagent according to the invention, are supplied in asample, are especially well-suited for determinations based on a changein the mass coverage of the solid carrier (support) (such as refractivemethods based on a change in the effective refractive index on thesurface of an optical waveguide, see below). For other applications itis advantageous to immobilize vesicles generated from living cells, froma bioanalytical reagent according to the invention, on a solid carrier(support) and then to address them (optionally in an individuallyaddressable way for different measurement areas) with one or moresamples comprising ligands to be detected. It is obvious to a personskilled in the art that embodiments analogous to those variants ofembodiments described above result from this inversion of the assayarchitecture.

It is characteristic of a large group of embodiments of thebioanalytical detection method according to the invention that one ormore liquid samples are brought into contact with the vesicles which aregenerated from living cells (from a bioanalytical reagent according tothe invention) and immobilized in one or more measurement areas, alongwith their associated receptors, and that a signal change resulting fromthe binding of ligands to said receptors or from other inducinginfluences on said receptors is measured.

Characteristic of a preferred embodiment is that one or more liquidsamples are brought into contact with the vesicles which are generatedfrom living cells (from a bioanalytical reagent according to theinvention) and immobilized in one or more measurement areas, along withtheir associated receptors, and that the signal transduction of thosereceptors resulting from the binding of ligands to said receptors orfrom other inducing influences on said receptors is measured.

For a special embodiment it is characteristic that the binding ofligands from a supplied sample to receptors that are associated with theimmobilized vesicles generated from living cells (from a bioanalyticalreagent according to any of claims 1-33) occurs in competition with thebinding of these ligands to receptors in free solution which areoptionally associated with vesicles.

Characteristic of a specially preferred embodiment is that one or moreliquid samples, comprising vesicles generated from living cells (from abioanalytical reagent according to the invention) with associatedreceptors, are brought into contact with the ligands for thesereceptors, the ligands being immobilized in one or more measurementareas, excitation light from one or more light sources of similar ordifferent wavelengths is in-coupled to the measurement areas (d) by oneor more grating structures (c), and the change of optical signalsemanating from one or more measurement areas (d), caused by a binding ofthe receptors associated with said vesicles to their immobilizedligands, is measured.

Characteristic of another specially preferred embodiment is that one ormore liquid samples are brought into contact with the vesiclesimmobilized in one or more measurement areas (d), along with theirassociated receptors, excitation light from one or more light sources ofsimilar or different wavelengths is in-coupled to the measurement areas(d) by one or more grating structures (c), and the change in opticalsignals emanating from one or more measurement areas (d), caused by thebinding of the ligands to said receptors or by other inducing influenceson said receptors, is measured.

These variants lead to various further possible embodiments of thebioanalytical detection method according to the invention.Characteristic of one group of embodiments is that said changes inoptical signals from the measurement areas (d) are caused by changes inthe effective refractive index in the near-field of the opticallytransparent layer (a) in these measurement areas and are measured at theactual excitation wavelength.

Characteristic of another preferred group of possible embodiments of thebioanalytical detection method according to the invention is that saidchanges in optical signals from the measurement areas (d) are changes inone or more luminescences of similar or different wavelength which havebeen excited in said measurement areas in the near-field of theoptically transparent layer (a), and which are measured each at awavelength different from the corresponding excitation wavelength.

It is preferred if the one or more luminescences and/or measurements oflight signals at the excitation wavelength are determinedpolarization-selectively, wherein preferably the one or moreluminescences are measured at a polarization that is different from thepolarization of the excitation light.

Subject of the invention in an again more general form is abioanalytical detection method according to any of the aforementionedembodiments for the simultaneous or sequential, quantitative and/orqualitative determination of one or more analytes from the group ofreceptors or ligands, chelators or “histidine tag components”, enzymes,enzyme co-factors or inhibitors.

It is characteristic of the bioanalytical detection method according toany of the aforementioned embodiments that the samples to be examinedare, for example, aqueous solutions or surface water or soil or plantextracts or bio- or process broths, or are taken from biological tissuefractions or from food, or odorous or flavoring substances or cosmeticcompounds.

A further subject of the invention is a solid carrier (support)comprising, immobilized on a surface, at least one vesicle generatedfrom a living cell, from a bioanalytical reagent according to theinvention and any of the aforementioned embodiments, said vesiclecomprising at least one receptor characterized by the fact that amechanism of signal transduction triggered by said receptor in the saidliving cell used for vesicle generation is preserved in said vesicleafter immobilization of the vesicle.

It is characteristic of a preferred embodiment of the solid carrier(support) according to the invention that vesicles, each comprising atleast one receptor, are immobilized in discrete measurement areas (d)with one or more vesicles each.

In this case, it is preferred if vesicles with at least two differentkinds of receptor are immobilized in multiple measurement areas (d),wherein vesicles with a uniform kind of receptor are preferablyimmobilized in each case within an individual measurement area.

The one or more vesicles generated from a living cell may be immobilizedon the surface of said solid support for example by means of covalentbinding or physical adsorption (electrostatic or van der Waalsinteraction or hydrophilic or hydrophobic interaction or a combinationof these interactions).

It is preferred if an adhesion-promoting layer is deposited between thesurface of said solid support and the one or more vesicles immobilizedthereon. According to the invention, the adhesion-promoting layer isdesigned in such a way that a mechanism of signal transduction triggeredby the one or more receptors in said living cell is preserved also afterimmobilization of the vesicles generated from a living cell, from abioanalytical reagent comprising at least one receptor, on saidadhesion-promoting layer.

The adhesion-promoting layer preferably has a thickness of less than 200nm, most preferably of less than 20 nm.

The adhesion-promoting layer may comprise a chemical compound of thegroup of silanes, epoxides, functionalized, charged or polar polymersand “self-organized functionalized mono or multiple layers”.

Characteristic of a preferred embodiment is that the adhesion-promotinglayer comprises a monomolecular layer of mainly one kind of protein,such as serum albumins or streptavidin, or of modified proteins, such asbiotinylated serum albumin.

Characteristic of another preferred embodiment is that theadhesion-promoting layer comprises self-organized alkane-terminatedmonolayers of mainly one kind of chemical or biochemical molecule.

Especially preferred is an embodiment of which it is characteristic thatthe adhesion-promoting layer is provided as a double layer (bilayer)comprising an initial self-organized alkane-terminated anchoring layerand a second layer formed by self-organization (self-assembly) ofsynthetic or natural lipids.

The one or more vesicles generated from a living cell may be immobilizedon the surface of said solid carrier (support), for example, by means ofcovalent binding or physical adsorption (electrostatic or van der Waalsinteraction or hydrophilic or hydrophobic interaction or a combinationof these interactions).

A specific embodiment of the solid carrier (support) according to theinvention, using a very specific variant of vesicle immobilization,comprises association with the adhesion-promoting layer of biological orbiochemical or synthetic recognition elements which recognize and bind avesicle generated from a living cell with surface-associated biologicalor biochemical or synthetic components for specific recognition andbinding, as part of the corresponding specific embodiment of abioanalytical reagent according to the invention described above. Thesespecific interactions for the recognition and binding of the vesicles totheir recognition elements on the adhesion-promoting layer may forexample be based on interactions with biotin/streptavidin, so-called“histidine tags”, sugars or peptide affinity interactions, wherein anyone of the two binding partners in each case may be associated with thevesicle surface and the other anchored on the surface of saidadhesion-promoting layer.

Characteristic of another embodiment of the solid carrier (support)according to the invention is that at least one ligand for a receptor,which is bound to a vesicle generated from a living cell, from abioanalytical reagent according to the invention, is immobilized,optionally by means of a spacer molecule, on the surface of the solidcarrier (support).

It is preferred in this case if at least two different ligands forreceptors which are bound to a vesicle generated from a living cell,from a bioanalytical reagent according to the invention, are immobilizedin multiple measurement areas (d), wherein preferably a uniform kind ofligand is immobilized within an individual measurement area.

Said ligands may be immobilized on the surface of the solid carrier(support) by means of covalent binding or physical adsorption (e.g.electrostatic or van der Waals interaction or hydrophilic or hydrophobicinteraction or a combination of these interactions).

It is preferred if an adhesion-promoting layer is applied between thesurface of the solid carrier (support) and said ligands immobilizedthereon.

For selection of the adhesion-promoting layer for immobilization of saidligands, the same preferences are applicable as those mentioned abovefor an adhesion-promoting layer for immobilization of vesicles generatedfrom a living cell, from a bioanalytical reagent according to theinvention.

Characteristic of a particularly preferred embodiment of a solid carrier(support) according to the invention is that regions between thelaterally separated measurement areas, with vesicles generated fromliving cells (from a bioanalytical reagent according to any of thedescribed embodiments) immobilized in these measurement areas, or withligands for receptors that are bound to vesicles generated from livingcells (from a bioanalytical reagent according to any of the describedembodiments), and/or regions within these measurement areas, between thecompounds immobilized therein, are “passivated” in order to minimizenonspecific binding of analytes or of their detection reagents, i.e.that compounds which are “chemically neutral” towards the analyte aredeposited between the laterally separated measurement areas (d) and/orwithin these measurement areas (d) between said immobilized compounds,the “chemically neutral” compounds preferably being composed of thegroups that are formed by albumins, casein, detergents, such as Tween20, detergent/lipid mixtures (of synthetic and/or natural lipids),synthetic and natural lipids or also hydrophilic polymers, such aspolyethylene glycols or dextrans.

It is also possible to passivate an activated surface (activated forimmobilization of the biological, biochemical or synthetic recognitionelements), this surface comprising e.g. poly-L-lysin or functionalizedsilanes (e.g. comprising aldehyde or epoxy groups), for example by theaddition of reducing reagents such as sodium borate (in the case ofaldehyde or epoxy groups).

The material of the surface of the solid support (carrier) withimmobilized vesicles generated from living cells (from a bioanalyticalreagent according to the invention and any of the describedembodiments), or with immobilized ligands for receptors that are boundto vesicles generated from living cells (from a bioanalytical reagentaccording to the invention and any of the described embodiments), maycomprise a material of the group which is formed e.g. by moldable,sprayable or millable plastics, carbon compounds, metals, such as gold,silver, copper, metal oxides or silicates, such as glass, quartz orceramics, or silicon or germanium or ZnSe or a mixture of thesematerials.

In this case, said solid carrier (support) may be provided in a varietyof different embodiments. It may be provided e.g. as a glass ormicroscope plate. It may also be a microtiter plate of the type that is,for example in widespread use for screening assays (for testing numerouscompounds, e.g. using classical fluorescence methods or fluorescencecorrelation spectroscopy).

It is preferred if the surface of said solid support (carrier) isessentially planar.

Characteristic of a preferred group of embodiments of the solid carrier(support) according to the invention is being provided as an optical orelectronic sensor platform.

Another subject of the invention is therefore a sensor platform as asolid support (carrier) according to any of the aforementionedembodiments, wherein said solid support is transparent at least in aregion of wavelengths in the ultraviolet to infrared spectrum andpreferably comprises a material from the group that is formed e.g. bymoldable, sprayable or millable plastics, carbon compounds, metals,metal oxides or silicates, such as glass, quartz or ceramics, or siliconor germanium or ZnSe or a mixture of these materials.

It is preferred in this case if said sensor platform is a solid carrier(support) wherein an optical waveguide serves as sensor platform.

Characteristic of a preferred embodiment of the sensor platform used asa solid carrier (support) is an optical thin-film waveguide serving as asensor platform, with an initial optically transparent layer (a) withrefractive index n₁ on a second optically transparent layer (b) withrefractive index n₂, wherein n₁>n₂.

For many applications such an embodiment of a sensor platform accordingto the invention used as solid carrier (support) is advantageous when itis divided into two or more discrete waveguiding regions.

The material of the second optically transparent layer (b) of the sensorplatform as a solid support may be selected from the group that isformed by silicates, such as glass or quartz, or transparent moldable,sprayable or millable, especially thermoplastic plastics, such aspolycarbonates, polyimides, polymethyl methacrylates, or polystyrenes.

It is preferred if the refractive index of the first opticallytransparent layer (a) of the sensor platform as a solid support isgreater than 1.8.

For numerous applications such an embodiment of a sensor platformaccording to the invention, used as a solid carrier (support), ispreferred when the first optically transparent layer (a) comprises amaterial of the group of TiO₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₂,preferably of TiO₂ or Ta₂O₅ or Nb₂O₅.

The first optically transparent layer (a) preferably has a thickness of40 to 300 nm, most preferably of 100 to 200 nm.

Characteristic of a further embodiment of the sensor platform accordingto the invention, used as solid carrier (support) is that an additionaloptically transparent layer (b′) with lower refractive index than layer(a) and with a thickness of 5 nm-10 000 nm, preferably of 10 nm-1000 nm,is located between the optically transparent layers (a) and (b) and incontact with layer (a).

It is preferred if the in-coupling of excitation light into theoptically transparent layer (a) to the measurement areas (d) isperformed using one or more optical in-coupling elements from the groupformed by prism couplers, evanescent couplers comprising joined opticalwaveguides with overlapping evanescent fields, butt-couplers withfocusing lenses, preferably cylindrical lenses, arranged in front of afront face (distal end) of the waveguiding layer, and grating couplers.

In this case, it is especially preferred if the in-coupling ofexcitation light into the optically transparent layer (a) to themeasurement areas (d) is performed using one or more grating structures(c) that are formed in the optically transparent layer (a).

Characteristic of an improvement of a sensor platform according to theinvention and any of the aforementioned embodiments is that theyadditionally comprise one or more sample compartments with said sensorplatform as the base plate, said sample compartments being open towardsthe sensor platform at least in the region of the one or moremeasurement areas, wherein said sample compartments may be open orclosed except for inlet and/or outlet openings at the side facing awayfrom the sensor platform.

A variety of further embodiments of sensor platforms, which are suitablein combination with a bioanalytical reagent according to the inventionand may be applied in a bioanalytical detection method according to theinvention, are described in detail, for example, in patents U.S. Pat.No. 5,822,472, U.S. Pat. No. 5,959,292, and U.S. Pat. No. 6,078,705, andin patent applications WO 96/35940, WO 97/37211, WO 98/08077, WO99/58963, PCT/EP 00/04869, and PCT/EP 00/07529. The embodiments ofsensor platforms and methods for the detection of one or more analytes,as well as the optical and analytical systems described therein, arealso a subject of the present invention, as part of sensor platformsaccording to the invention, as solid carriers. (supports) comprising abioanalytical reagent according to the invention, and as parts ofbioanalytical detection methods according to the invention which areperformed therewith.

A further subject of the present invention is the use of a vesicle as acomponent of a bioanalytical reagent according to the invention and anyof the aforementioned embodiments and/or of a solid carrier (support)according to the invention, comprising one or more vesicles immobilizedthereon, as described in any of the aforementioned embodiments for theenrichment of membrane receptors or for the enrichment of proteins (suchas antigens) triggering an immunological response in a two- orthree-dimensional phase, which may then e.g. be administered to livingorganisms (e.g. to stimulate immune defense processes).

A further subject of the invention is the use of a vesicle as acomponent of a bioanalytical reagent according to the invention, asdescribed in any of the aforementioned embodiments, as a compartment fortherapeutic, diagnostic, photosensitive or other biologically activecompounds for administration to a living organism.

The present invention also comprises the use of a bioanalytical reagentaccording to the invention as described in any of the aforementionedembodiments and/or of a solid carrier (support) according to theinvention as described in any of the aforementioned embodiments,comprising one or more immobilized vesicles, and/or of a bioanalyticaldetection method according to the invention as described in any of theaforementioned embodiments for investigating receptor-ligandinteractions, especially for determining the binding strength andkinetic parameters of these interactions between a receptor and itsligand, or for determining the channel activity of an ion channelreceptor after ligand binding or other inducing influences on saidreceptor, or for determining the enzymatic activity of enzymesassociated with a vesicle, as a component of a bioanalytical reagentaccording to the invention, or for determining secondary messengercompounds after ligand binding to a receptor resulting in a signaltransduction, or for determining protein-protein interactions, or fordetermining protein kinases.

The present invention additionally comprises the use of a bioanalyticalreagent according to the invention as described in any of theaforementioned embodiments and/or of a solid carrier (support) accordingto the invention as described in any of the aforementioned embodiments,comprising one or more immobilized vesicles, and/or of a bioanalyticaldetection method according to the invention as described in any of theaforementioned embodiments for quantitative and/or qualitative analysesfor determining chemical, biochemical or biological analytes inscreening methods in pharmaceutical research, combinatorial chemistry,clinical and pre-clinical development, for real-time binding studies andfor determining kinetic parameters in affinity screening and inresearch, for qualitative and quantitative analyte determinations,especially for DNA and RNA analytics, for generation of toxicity studiesand for the determination of expression profiles, and for determiningantibodies, antigens, pathogens or bacteria in pharmaceutical productdevelopment and research, human and veterinary diagnostics, agrochemicalproduct development and research, for symptomatic and pre-symptomaticplant diagnostics, for patient stratification in pharmaceutical productdevelopment and for therapeutic drug selection, for determiningpathogens, nocuous agents and germs, especially of salmonella, prionsand bacteria, in food and environmental analytics, and for analysis andquality control of odorous and flavoring substances.

EXAMPLES

The present examples describe the preparation of a bioanalytical reagentaccording to the invention with at least one vesicle which has beengenerated from a living cell, comprising at least one receptor, whereina mechanism of signal transduction triggered by this receptor in theliving cell used remains preserved in said vesicle as part of thebioanalytical reagent.

In these examples, it is shown

-   -   that membrane receptors of membranes of living cells can be        transferred to membranes of “native” vesicles without loss of        function    -   that components from the lumen of the vesicle-forming cell can        be transferred to the “native” vesicles formed without an        exchange with the surrounding medium    -   that luminescence indicators required for secondary responses        can be incorporated into “native” vesicles    -   and that the transferred components including the indicators        remain stable within the interior of “native” vesicles.

Example 1 Preparation of “Native” Vesicles as Component of aBioanalytical Reagent According to the Invention and Visualization ofthe Prepared Vesicles

1.1. Preparation of Vesicles of Defined Size

For the preparation of vesicles (“vesiculation process”) adherentgrowing HEK293 (human embryonic kidney) cells were cultured in each casein 15 ml DMEM/F12 (Dulbecco's modified Eagle medium, Gibco BRL LifeTechnologies) in 75 ml T flasks (TPP, Switzerland). To the medium, 2.2%fetal calf serum (Gibco BRL Life Technologies) was added. The cellcultures were stored in an incubator (37° C., 5% CO₂).

To visualize the cytoplasmic contents of the cell during thevesiculation process, HEK293 cells were transfected with plasmid DNAs(Clontech; Palo Alto, Calif., USA) coding for Aequorea victoria GFP(Green Fluorescent Protein) using the customary method of calciumphosphate-DNA coprecipitation (Jordan M., Schallhorn A., Wurm F. M-:“Transfecting mammalian cells: optimization of critical parametersaffecting calcium-phosphate precipitate formation”, Nucleic Acids Res.24 (1996) 596-601). Twenty hours after transfection, the greenfluorescence of GFP was visible in the entire cell contents, afterexcitation at 488 nm, using a confocal fluorescence microscope. Thiscontrol procedure was used in particular to demonstrate that, during thevesiculation process, the cell and vesicle content remains intact, i.e.that there is no contact and no intermixing with the surrounding outercell medium (see FIG. 1).

For vesiculation, the serum contained in the culture medium was removedby decantation or, in the case of the suspension cells, bycentrifugation (rotor SS34 Sorvall, at 120 g; centrifugation time 5minutes) and exchanged for serum-free DEMEM medium. The cellconcentration lay between 1×10⁷ and 5×10⁷ cells per milliliter.

Cytochalasin B or D (Sigma-Aldrich) (stock solution with a concentrationof 2 mg/ml in DMSO) was added to the preheated DMEM medium in a finalconcentration of 20 μg/ml. The vesiculation process was dependent on thecell age and cell line and lasted between 5 and 60 minutes. Shear forceswere then applied (1 minute vortex) to separate off any vesiclesremaining on the cell surface as completely as possible. This suspensionwas then passed through a sterile syringe filter (5 μm pore size;Acrodisc) to separate off any remaining cell bodies from the vesicles.The vesicle suspension was then concentrated by centrifugation (rotor SS34, Sorvall, at 5400 r.p.m. (revolutions per minute; 20 minutes).

The resulting vesicle pellet was then resuspended in 500 μl phosphatebuffer (pH 7.5) and loaded onto a preformed sucrose gradient. Thisgradient comprised three sucrose layers with decreasing density (inascending order 2 M, 1.5 M, 1.3 M sucrose in deionized, sterile water).After 90 minutes of centrifugation at 25000 r.p.m. in a centrifuge rotor(TST 60.4 at 84,840 g; Sorvall; Kontron ultracentrifuge) three clearlyseparated bands were visible. The bands contained vesicles of varioussizes. The vesicle membranes were visualized by staining with octadecylrhodamine B chloride (R18) (Molecular Probes Inc., USA) in order tomeasure the size of the vesicles in a confocal fluorescence microscope(excitation: 560 nm/emission: 590 nm) using this fluorescence labeling.The uppermost band contained vesicles with a diameter of 100-300 nm, themiddle band contained vesicles with a diameter of 500-800 nm and thelowest band contained vesicles with a size distribution between 1 μm and3 μm.

The “native” vesicles produced in this way were thus smaller than, forexample, red blood cells (3-5 μm). The vesicles from the two upper bandswere even smaller than mitochondria.

As shown in the following, the “native vesicles” contain parts of theendoplasmic reticulum. In view of their size, however, the cell nucleusand mitochondria (in vesicles with a diameter of less than 800 nm) areexcluded from incorporation into the vesicles. In the case ofG-protein-coupled receptors (GPCR) it follows from this sizedistribution, for example, that the vesicles can only containendoplasmic reticulum, albeit as the most important reservoir ofintracellular calcium ions and as essential component of the GPCR-signaltransduction cascade.

Vesicles from the uppermost band were used for experiments aimed atinvestigating the residual capacity of receptors for ligand bindingfollowing preparation, whereas vesicles from the two lower fractionswere used for detecting the release of secondary messenger compounds,such as Ca²⁺ and cAMP, because these vesicles can be charged to agreater extent with suitable fluorescence indicators.

The process of budding and pinching of cells in vesicle formation isschematically illustrated in the diagram below the fluorescencemicroscopy images (FIG. 1): (a) Normal actin filament network in cellcortex. (b) The actin filaments retract at certain points for someminutes after administration of cytochalasin B/D (concentration 20μg/ml), and the endoplasm of the cell can expand locally, resulting in(c) cell budding and pinching.

1.2. Determination of Endoplasmic Reticulum in “Native” Vesicles

For determination of the endoplasmic reticulum in native vesicles, as aleading precondition for the release of secondary Ca²⁺ after activationof the signal transduction cascade of G-protein-coupled receptors, thegreen fluorescent protein (GFP) of Aequorea Victoria at the level of thecoding DNA was furnished with a peptide signal sequence(MRLCIPQVLLALFLSMLTAPGEG) which, during the synthesis of GFP in thecell, guides it into the endoplasmic reticulum. This molecularbiological intervention did not have any negative influence on thevitality of the cultivated HEK293 cells. The overlapping of GFPfluorescence with the geometric dimensions of the endoplasmic reticulum(ER) was studied using commercial lipophilic tracer for ER(1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(DiIC16(3); Molecular Probes) in whole cells. Based on the fluorescenceof the GFP molecules, parts of the endoplasmic reticulum weredemonstrated in native vesicles using confocal microscopy images (FIG.2).

1.3. Preparation and Determination of “Native” Vesicles with (A)Incorporated, Fluorescence-Labeled G-Protein and (B) IncorporatedIndicators

(A) “Native” vesicles were prepared with the recombinant G-alpha subunitof a G-protein (G_(α)). To demonstrate the presence of this protein inliving HEK293 cells using a confocal fluorescence microscope, theG-alpha-15 protein (G_(α15)) at the level of the coding DNA was fusedwith Aequorea Victoria GFP. For this purpose the DNA coding for EGFP(Enhanced Green Fluorescent Protein; Clontech) was inserted in theG_(αq) subunit. The resulting fusion protein was transfected into HEK293cells using the above-mentioned calcium phosphate precipitation method.24 hours after transfection, the green-labeled G_(α)-protein wasdetectable in the cytoplasm (as in FIG. 1 a of Example 1.1). Therecombinantly expressed proteins were localized close to the cellmembrane using confocal microscopy.

After cytochalasin B (2 mM) was added to the HEK293 cells expressing Gprotein, plasma membrane vesicles which had incorporated greenfluorescent G_(α) fusion protein were pinched off.

(B) “Native” vesicles were prepared with incorporated, chemicalfluorescence indicators. For this purpose, HEK cells were loaded withcalcium-sensitive indicators (Fura Red, C₄₇H₅₂N₄O₂₄S, 149732-62-7glycine,N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-N-[5-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]-2-[(5-oxo-2-thioxo-4-imidazolidinylidene)methyl]-6-benzofuranyl]-,(acetyloxy)methyl ester and Fluo-3/AM (C₃₆H₄₅Cl₂N₇O₁₃; 121714-22-5glycine,N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-,(acetyloxy)methyl ester; Molecular Probes). Per milliliter of cellnutrient culture medium (DMEM/F12: Dulbecco's modified Eagle Medium F12)12.5 μg Fluo-3/AM and 24.5 μg Fura Red were added. The ion-sensitiveindicators were dissolved beforehand in DMSO. Loading with acetoxymethylester lasted 60 minutes at 37° C. Addition of cytochalasin led topinching-off of vesicles with a marked indicator load. Both indicatorswere clearly visible under fluorescence microscopy. The concentration offree Ca 2+ions in the interior of the vesicles and also theconcentration of free Ca²⁺ ions in the cytosol of the cell from whichthey originated were determined by analyzing the measured fluorescenceintensities. The two were comparable and amounted to 130 nM.

1.4. Preparation of Vesicles with Incorporated, Recombinant FusionProteins from G Proteins and Fluorescence-Labeled Proteins as well asIncorporated Indicators

The DNA coding for EGFP (Enhanced Green Fluorescent Protein; Clontech)was inserted in the G_(αq) subunit. The resulting fusion protein wastransfected into HEK293 cells using the above-mentioned calciumphosphate precipitation method. 24 hours after transfection, the greenlabeled G_(α) protein was detectable in the cytoplasm and showed, inaddition to the occurrence of diffuse areas, a frequent tendency towardsthe formation of aggregates (0.5-1 μm in diameter) close to the plasmamembrane. After cytochalasin B (2 mM) was added to the HEK293 cellsexpressing G protein, plasma membrane vesicles which had incorporatedthe green fluorescent G_(α) fusion protein were pinched off (FIG. 3).The cells were loaded with calcium-sensitive indicators (Fura Red andFluo-3/AM). For every milliliter of cell nutrient culture medium(DMEM/F12: Dulbecco's modified Eagle Medium F12) 12.5 μg Fluo-3/AM and24.5 μg Fura Red were added. The ion-sensitive indicators were dissolvedbeforehand in DMSO. Loading with acetoxymethyl ester lasted 60 minutesat 37° C. Addition of cytochalasin led to pinching-off ofindicator-loaded vesicles. Both indicators were clearly visible in thevesicle lumen on fluorescence microscopy (FIG. 3). Analysis of themeasured fluorescence intensities showed a concentration of free Ca²⁺ions amounting to 130 nM, as is also measured in the determination offree Ca²⁺ ions in the cytosol of a cell.

Example 2 Preparation of “Native” Vesicles Capable of SignalTransduction with Incorporated Receptors and the Detection Thereof

2.1. Incorporation of Ion Channel Receptors in “Native” Vesicles withthe Preservation of Their Functionality, as Illustrated in the 5HT_(3A)Receptor

Inn the following examples, the 5HT₃ serotonin receptor is used as arepresentative ligand-controlled ion channel. In the literature, twodifferent types of 5HT₃ receptor are described, the 5HT_(3A) and the5HT_(3B) receptor (Davies P. A., Pistis M., Hanna M. C., Peters J. A.,Lambert J. J., Hales T. G., Kirkness E. F., “The 5-HT_(3B) subunit is amajor determinant of serotonin-receptor function”, Nature 397 (1999)359-363). In the following examples, only the 5HT_(3A) receptor is used.

Various recombinant constructs of the serotonin (5HT_(3A)) receptor wereexpressed in HEK293 cells under human cytomegalovirus gene promotercontrol. Transient expression of the full wild-type receptor wasachieved by co-transfection of the eukaryotic expression vector CMVβ(Clontech, Palo Alto, Calif.) with the receptor of coding cDNA and/orwith corresponding cDNA for cytosolic GFP, in order to identify cellswhich expressed the serotonin receptor (5-HT_(3A) R) at the same time.

16 to 20 hours before transfection, HEK293 cells (10⁵ cells/ml) wereseeded in 6-well plates or, in the case of samples for laterinvestigation under confocal fluorescence microscopy, on sterile coverglasses (diameter 22 mm) in 6-well plates. The cells were transfectedusing Effectene (Qiagen, Hilden, Germany) in accordance with themanufacturer's instructions. After four hours' transfection in a humidatmosphere (5% CO₂, 37° C.) the transfection medium was exchanged withfresh cell culture medium.

Vesicles were prepared from the cells 48 hours after transfection in amanner similar to that described in Example 1.1. For this purpose, thecells were exposed to a trypsin-EDTA solution (Sigma) in two 6-wellplates (0.5-1 ml per well) for one to two minutes with gentle stirring.The well contents were then centrifuged for 5 minutes (1200 r.p.m.,Rotor SLA-600, Sorvall, Newtown, USA). The supernatant was discarded andthe resulting pellet resuspended in 10 ml in PBS buffer (10 mM phosphatebuffer solution with Na₂HPO₄, K₂HPO₄, 138 mM NaCL, 2.7 mM KCl, pH 7.4)in a vortex mixer. The generation of vesicles from the cell preparationwas triggered by the addition of cytochalasin from a stock solution inDMSO (4 mg/ml) up to a final concentration of 20 μg/ml. The resultingpinched-off vesicles were separated from the cells by shear forcesapplied again by means of a vortex (2 minutes), before the suspensionwas finally resuspended and the supernatant then passed through asyringe filter (1.2 μm pore size, Acrodisc) with the vesicles containedtherein.

According to the above description, “native” vesicles were prepared withexpressed receptor contained therein. The presence of the 5HT_(3A)receptor in the outer vesicle membrane was detected by labeling of thisreceptor with the receptor-specific ligand GR-Cy5(=1,2,3,9-tetrahydro-3-[(5-methyl-1h-imidazol-4-yl)methyl]-9-(3-amino-(N-Cy5-amide)-propyl)-4H-carbazol-4-one).Intensity profiles of the fluorescence from fluorescence-labeledreceptors and of GFP, taken up by the vesicles from the GFP-labeledcytosolic cell contents, unequivocally demonstrate the existence of thereceptor in the “native” vesicle membrane and thus its origin from theplasma membrane of the vesicle-forming mammalian cell. The presence ofGFP in the “native” vesicle unequivocally demonstrates the cytosolicorigin from the cell (FIG. 4).

2.1.1 Determination of Ligand-Binding Capacity of the 5HT_(3A) Receptorin “Native” Vesicles in Solution

The capacity of 5HT_(3A) receptors for specific ligand binding, as their“primary” functionality, after incorporation of the receptors in“native” vesicles was investigated by means of a competitiveradiological binding assay (Tairi A. P., Hovius R., Pick H., Blasey H.,Bernard A., Surprenant A., Lundstrom K., Vogel H., “Ligand binding tothe serotonin 5HT₃ receptor studied with a novel fluorescent ligand”,Biochemistry 37 (1998) 15850-15864; Wohland T., Friedrich K., Hovius R.,Vogel H., “Study of ligand-receptor interactions by fluorescencecorrelation spectroscopy with different fluorophores: evidence that thehomopentameric 5-hydroxytryptamine type 3As receptor binds only oneligand”, Biochemistry 38 (1999) 8671-8681).

To this end, samples of said reagent were incubated with the vesicles insolution for 60 minutes at room temperature with 1.5 nM of thetritium-labeled ligand [³H]-GR65630 in 240 μl HEPES buffer (10 mM HEPES,pH 7.4) (HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) in96-well MultiScreen plates (Millipore, F-Molsheim). The incubation wascompleted by rapid filtration followed by washing 3 times with 300 μl ofice-cold HEPES buffer each time. The filters were transferred toscintillation vessels and each taken up in 1 ml of Ultima Gold TM(Packard, meridan, USA). The radioactivity was measured using a TriCarb2200CA liquid scintillation counter. The extent of nonspecific bindingto the receptor and to the vesicles was estimated in the presence of ahigh surplus of quipazine (1 μM). The dissociation constant K_(d) wasdetermined from Scatchard plots in 6 different concentrations of theligand [³H]-GR65630. The binding behavior (“pharmacology”) of thereceptor was determined in the form of IC₅₀ values (concentration at 50%inhibition) through the competition of various pharmacologically activesubstances with the binding of the radioactively labeled referenceligand [³H]-GR65630. All experiments were carried out in duplicate. Forcomparison purposes, experiments were also conducted under comparableconditions in the whole mother cells. The following table summarizes theexperimentally determined dissociation constants. Receptor in Receptormother cell in “native” vesicles pK_(i) pK_(i) Antagonist GR-H 10.2 ±0.1  9.7 ± 0.1 Granisetron 9.2 ± 0.1 9.5 ± 0.1 Ondansetron 8.5 ± 0.1 8.6± 0.1 Agonist Quipazine 9.3 ± 0.1 9.4 ± 0.2 mCPBG 8.2 ± 0.1 8.3 ± 0.15HT 7.5 ± 0.1 7.7 ± 0.1 PBG 6.5 ± 0.1 6.6 ± 0.1 [³H]-GR65630 9.0 ± 0.19.4 ± 0.1

Explanation of abbreviated names: Antagonist GR-H1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl)]-9-(3-aminopropyl)-4H-carbazol-4-one Granisetron:endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methyl-1H-indazole-3-carboxamide hydrochloride Ondansetron:1,2,3,9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one Agonist: Quipazine:2-(1-Piperazinyl)quinoline m-CPBG: m-Chlorophenylbiguanide 5-HT:5-Hydroxytryptamine PBG: Phenylbiguanide [³H]-GR65630 see: Kilpatrick G.J., Jones G. J., Tyers M. B., (1988) “The distribution of specificbinding of the 5-HT3 receptor ligand [³H]-GR65630 in rat brain usingquantitative autoradiography”, Neuroscience Letters 94 (1988) 156-160.2.1.1 Determination of Ligand-Binding Capacity of the 5HT_(3A) Receptorin Surface-Immobilized “Native” Vesicles

The ligand-binding experiments in surface-immobilized vesicles werecarried out using the fluorescence-labeled ligand GR-Cy5. For theseexperiments, a homologous serotonin receptor construct 5HT_(3A) was usedthat was expressed with an EGFP (Enhanced Green Fluorescent Protein,Clontech) fused at the end of a subunit. The prepared “native” vesicleswere immobilized on cover glasses by means of physical adsorption of thevesicles to the glasses, by two-hour or overnight incubation oftypically 400 μl of a vesicle preparation solution prepared according tothe above method in 6-well plates with cover glasses on the bottom at 4°C. The cover glasses were then removed from the plates with the vesiclesimmobilized thereon and inserted into an open cell, which was thenfilled with 200-300 μl PBS buffer.

The affinity of a fluorescent ligand for the vesicle-bound 5HT_(3A)receptors, in an experiment with a large number of vesicles, was studiedby incubating a series of cover glasses for 2 hours each with vesiclesin solution and increasing concentrations of fluorescent ligands at roomtemperature. A second series of cover glasses was incubated under thesame conditions, for estimating the extent of nonspecific binding of thefluorescent ligands to the vesicles, likewise with vesicles in solutionand the same increasing concentrations of fluorescent ligands, as wellas 5 μM quipazine, as competitor in a high (>150-fold) surplusconcentration.

The affinity of the fluorescent ligand for receptors bound toindividual, discrete vesicles was determined by sequential addition ofascending concentrations of the fluorescent ligand to one and the samecover glass with vesicles immobilized thereon by overnight incubation.The studies were carried out in a manner analogous to that describedabove for a large number of vesicles.

The fluorescence intensities of the ligand were measured with a confocalfluorescence microscope (Zeiss, Laser Scanning Microscope LSM510), usinga suitable filter set. The fluorescence signal of the GFP additionallyincorporated into the vesicles was used in each case to adjust themicroscope to the working distance of the plane in which the vesicleslay before the ligand was added. The fluorescence signals of the ligands(red) were referenced via the fluorescence signals of the EGFP (green)according to the number of active receptors per vesicle. The imagespresented were obtained from the signals of the fluorescence microscoperecorded with a photomultiplier. For the image analysis, so-called“Regions of Interest” (ROIs) of the images were defined, the dimensionsof which were adjusted to the areas to be measured.

The results of this experiment show that vesicles show differing numbersof associated 5HT_(3A) receptors, with the consequence of markedlydiffering fluorescence intensities of different vesicles, but thebinding constants showed identical values in agreement with the valueswhich have been reported in the literature and which were determinedwith isolated receptors or in complete cells (Tairi A. P., Hovius R.,Pick H., Blasey H., Bernard A., Surprenant A., Lundstrom K., Vogel H.,“Ligand binding to the serotonin 5HT₃ receptor studied with a novelfluorescent ligand”, Biochemistry 37 (1998) 15850-15864; Wohland T.,Friedrich K., Hovius R., Vogel H., “Study of ligand-receptorinteractions by fluorescence correlation spectroscopy with differentfluorophores: evidence that the homopentameric 5-hydroxytryptamine type3As receptor binds only one ligand”, Biochemistry 38 (1999) 8671-8681).

As an example, FIG. 5 shows the measured fluorescence intensities as afunction of increasing concentrations of GR-Rho(1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-rhodamineB-thiocarbamoyl)-propyl)-4H-carbazol-4-one)as fluorescent ligand (after subtraction of the fluorescence intensityF₀ observed in the absence of GR-Rho). The fit of measurement data withthe Langmuir isotherm equation gave a value of K_(d)=(4.2+/−1.7) nM forthe dissociation constant of the system comprising the HT₃ receptor andthe GR-Rho ligand (FIG. 5).

2.1.3. Functional Capacity of the Full Signal Transduction Cascade of aReceptor Incorporated in “Native” Vesicles: Induction of Ion-ChannelActivity (Secondary Function) of the 5HT_(3A) Receptor after SpecificLigand Binding (Primary Function).

It was surprisingly found that, with a bioanalytical reagent accordingto the invention, the full signal transduction transmitted via areceptor is preserved.

FIG. 6 demonstrates the ability of vesicles with incorporated 5HT_(3A)serotonin receptor to regulate the concentration of intracellularcalcium. In this case, the original HEK cells from which the vesicleswere obtained following the addition of cytochalasin according to themethod described hereinabove were loaded with the acetoxymethyl ester ofFluo-3 as calcium indicator (generally 20 μg Fluo-3 in 500 μl DMEM/F12medium). The prepared vesicles contained said fluorescent calciumindicator and parts of the endoplasmic reticulum in addition to the5HT_(3A) receptor.

Fluorescence excitation of the Fluo-3 was performed at 488 nm. Theemission was measured at 500-530 nm. The vesicles loaded with Fluo-3were then stimulated with 350 μM m-chlorophenylbiguanide hydrochloride(mCPG), a receptor-specific agonist. Stimulation was performed byaddition of the agonist to the vesicle medium (t=0 sec). The maximumfluorescence signal that occurred through binding of the calcium to thecalcium indicator Fluo-3 was already measured less than one minute afteraddition of the agonist. The value amounted to 35 μM free calcium ionsin the vesicle interior, i.e. in the cytosol surrounded by the vesicle.With the aid of calcium indicator Fluo-3, free calcium ions are detectedonly in the cytosol (vesicle lumen) transferred from the cell of origin.Calcium ions in the endoplasmic reticulum or in other cell organelles orparts of cell organelles possibly included in the vesicle are notdetermined by this detection method.

The increase in the fluorescence signal up to its maximum value afterless than one minute and the subsequent decrease in the fluorescencesignal to its baseline value after a further 2 minutes, i.e. after atotal of 3 minutes following addition of the agonist, is a sign of theintact functional capacity of the complete mechanism of the signaltransduction cascade within the vesicle, i.e. of the ability of thevesicle to “buffer” the concentration of free calcium ions. Thisincludes the action of sodium-calcium exchangers, in addition to thetimespan until closure of the receptor's ion channel and desensitizationmechanisms characteristic of serotonin.

2.2. Incorporation of G-Protein-Coupled Receptors in “Native” Vesicleswith the Preservation of Their Functionality, as Illustrated in the NK1Receptor

An important member of the membrane-bound G-protein-coupled receptors(GPCRs) is the NK1 receptor. It plays an important immunological role inthe activation of astrocytes in the central nervous system by substanceP, a tachykinin. Tachykinins are neuropeptides which are active both inthe peripheral and in the central nervous system and play a role ininflammatory processes, nociception and a number of autonomic reflexes.

There is evidence to suggest that GPCRs induce membrane ruffling(Okamoto H., Takuwa N., Yokomizo T., Sugimoto N., Sakurada S.,Shigematsu H., Takuwa Y., “Inhibitory Regulation of Rac Activation,Membrane Ruffling, and Cell Migration by the G Protein-CoupledSphingosine-1-Phosphate Receptor EDG5 but Not EDG1 or EDG3”, Mol CellBiol 20 (2000) 9247-9261). This different behavior compared with that ofthe 5HT3 receptor described could influence incorporation in the nativevesicles as a deleterious reagent. The following shows that this is notthe case.

The NK1 receptor was fused with the DNA coding for EGFP (Clontech PaloAlto, Calif., USA) at the level of the coding DNA on thecarboxy-terminal end and transfected into HEK293 cells by means of thecalcium-phosphate/DNA co-precipitation method (Jordan M., Schallhorn A.,Wurm F. M., “Transfecting mammalian cells: optimization of criticalparameters affecting calcium-phosphate precipitate formation”; NucleicAcids Res 24 (1996) 596-601). 24 hours after transfection, green labeledNK1 receptors were detectable in the plasma membrane of the HEK293cells. Cytochalasin B (10 μg/ml) was then added to the expressing cellsand vesicle formation observed under confocal microscopy. FIG. 7demonstrates the incorporation of NK1-GFP fusion proteins in “native”vesicles. Using the NK1 receptor as a example, evidence is thus shownthat G-protein-coupled receptors are incorporated in native vesicles.

3. Storage Life of Native Vesicles

Freshly prepared “native” vesicles could be stored for up to one week inphosphate buffer (PBS: 150 mM sodium phosphate, 150 mM NaCl, pH 7.2+0.1(25° C.) or DMEM/F12 medium) under sterile conditions at 4° C. withoutany change in quality.

For freezing, “native” vesicles were resuspended in DMEM medium with2.2% serum (fetal bovine serum) and 10% dimethylsulfoxide (DMSO). Thisvesicle suspension was shock-frozen in liquid nitrogen. Frozen vesiclescould be stored up to 6 months at −80° C. without loss of quality.Compared with a freshly prepared vesicle fraction, frozen “native”vesicles did not show any significant changes in size or morphologyafter they were thawed out. They also did not show any increasedtendency towards aggregation formation.

Determinations on the Stability of Frozen Vesicles:

Vesicles were prepared from HEK293 cells which expressed the GFP ofAquorea victoria in their cytoplasm. A fluorescent protein freelydissolved in cytoplasm should serve as evidence to show that thevesicles survive the freezing and thawing cycle intact and do not burstor drain. FIG. 8 shows an example of microscopy images of vesicles after30 days' storage at −80° C. No damage or change to the morphology of thevesicles was observed.

DESCRIPTION OF FIGURES

FIG. 1: Process of vesicles being pinched off with the transfer ofcytoplasm from the cell into the vesicles: Images produced with aconfocal fluorescence microscope showing HEK293 cells which, aftertransfection with Aequorea GFP, display a green fluorescence in thecytoplasm (excitation 488 nm/emission 510 nm); below, graphicillustrations of the process. (a) Normal actin-filament network in thecell cortex before the addition of cytochalasin. (b) After applicationof cytochalasin B, the cells begin to round off and form (c) bullous orpedunculate buds.

FIG. 2: Confocal image (excitation: 488 nm; emission: 510 nm)illustrating the incorporation of parts of the endoplasmic reticuluminto “native” vesicles ((a) image at the fluorescence wavelength; (b)image at the fluorescence wavelength superimposed on transmissionimage). The endoplasmic reticulum was visualized by the expression ofrecombinant EGFP (Enhanced GFP; Clontech, Palo Alto, Calif., USA) inHEK293 cells.

The EGFP here was furnished with a signal sequence which guides thefluorescent protein during its expression into the lumen of theendoplasmic reticulum (ER), thus “staining” the ER. The arrow marks avesicle that was pinched off from a living HEK293 cell after theaddition of cytochalasin B [10 μg/ml]. The bar bottom right correspondsto a length of 5 μm.

FIG. 3: Confocal image of a dividing HEK cell treated for vesicleformation, which expresses heterologously GFP-labeled G_(α) protein. Thecell had been treated beforehand with the calcium indicator Fura Red.Shown here in clockwise order, starting top left, are: (a) Fura Redsignal, (b) GFP-G_(α) signal, (c) transmission image, (d)superimposition of images (a) and (c). The Fura Red signal (image (a))was recorded using a high-pass filter (cut-off filter with transmissionfor λ>650 nm). Image (b) shows the fluorescence emission of the GFPbetween 500 and 530 nm. The transmission image corresponds to aNORMARSKI image. For all the images shown, fluorescence excitation tookplace at 488 nm.—“Native” vesicles, budding vesicles and those whichhave already pinched off are shown with circles. Note that the vesiclesare carrying both the recombinant product (Ga protein) and the calciumindicator.

FIG. 4: (a) Fluorescence of a vesicle produced from a living HEK293 cellwith GFP-labeled cytosolic cell contents carried over from the cell oforigin (green fluorescence in vesicle interior) and 5HT3 receptorincorporated in the vesicle membrane, labeled with GR-Cy5 (redfluorescence from the outer region of the vesicle). (b) Line profiles ofgreen and red fluorescence.

FIG. 5: Binding curve of a fluorescent ligand bound to individualdiscrete vesicles (n=3) was determined by sequential addition ofascending concentrations of the fluorescent ligand to one and the samecover glass with vesicles immobilized thereon by overnight incubation.

Fluorescence intensities F as a function of ascending concentrations ofGR-Rho, presented as fluorescent ligand of the 5HT₃ receptorincorporated in “native” vesicles from HEK293 cells (after subtractionof the fluorescence intensity F₀ in the absence of GR-Rho). The fit ofmeasurement data with the Langmuir binding isotherm equation gives avalue of K_(d)=(4.2+/−1.7) nM for the dissociation constant of thesystem comprising the HT₃ receptor and the GR-Rho ligand.

FIG. 6: Demonstration of the ability of the serotonin 5-HT₃ receptorincorporated in vesicles to regulate the concentration of intravesicularcalcium ions, visualized on the basis of the fluorescence of the calciumindicator Fluo-3 (excitation: 488 nm; emission: 500-530 nm). Stimulationof vesicles loaded with Fluo-3 by addition of 350 μMm-chlorophenylbiguanide hydrochloride (mCPG), as a receptor-specificagonist.

Confocal false-color images before and t=0 (a, b), 45 sec (c) and 3 min(d) after addition of the serotonin agonist. Shot (b) presents theanalyzed image sections (circles) against the background of thetransmission image, superimposed with the fluorescence of the calciumindicator not coded with false colors. Note that the fluorescencesignals encompass a larger area than the vesicles in view of the highdegree of enhancement.

(e) Time curve of the fluorescence signal of a vesicle which showed thestrongest Ca response. The stimulation was performed by addition of theagonist (mCPG) to the vesicle medium (t=0 sec). The maximum fluorescencesignal that occurred through binding of the calcium to the calciumindicator Fluo-3 was already measured after less than one minute.

FIG. 7: A “native” vesicle becoming detached from an HEK293 cell, whichexpresses the GFP-labeled NK1 receptor. The NK1 receptor protein herewas fused with the Aequorea victoria GFP at the level of the coding DNA.

FIG. 8: Confocal images illustrating the quality of “native” vesicleswhich have been stored for 30 days at −80° C. and then thawed out.“Native” vesicles were produced by cytochalasin B treatment of HEK293cells which express the transiently transfected, recombinant EGFP(Clontech; Palo Alto, Calif., USA) in the cytosol. (a) Fluorescenceimage (excitation 488 nm; emission 510 nm); (b) combination oftransmission image and fluorescence image. The section at bottom rightshows a greater magnification of intact “native” vesicles.

1-112. (canceled).
 113. A bioanalytical reagent with at least onevesicle, generated from a living cell, comprising at least one receptor,characterized in that a mechanism of signal transduction triggered bysaid receptor in the cell used for vesicle generation is preserved insaid vesicle as a component of the bioanalytical reagent.
 114. Abioanalytical reagent with at least one vesicle, generated from a livingcell, according to claim 113, wherein the vesicle as a component of saidreagent comprises further cell products and/or cell proteins, besidessaid one or more receptors, which are involved in said mechanism ofsignal transduction, besides said one or more receptors.
 115. Abioanalytical reagent with at least one vesicle, generated from a livingcell, according to claim 113, wherein said one or more vesicles aregenerated from a eukaryotic cell.
 116. A bioanalytical reagent with atleast one vesicle, generated from a living cell, according to claim 113,wherein said one or more vesicles are generated from a cell of a nativetissue.
 117. A bioanalytical reagent with at least one vesicle,generated from a living cell, according to claim 113, wherein theinterior of said one or more vesicles is free from cell nucleusmaterial, so that replication processes do not occur within said one ormore vesicles.
 118. A bioanalytical reagent with at least one vesicle,generated from a living cell, according to claim 113, wherein said oneor more vesicles have a diameter of 50 nm-5000 nm, preferably of 100nm-2000 nm.
 119. A bioanalytical reagent with at least one vesicle,generated from a living cell, according to claim 113, wherein the one ormore receptors are present in natural form in said one or more vesiclesas a component of the bioanalytical reagent.
 120. A bioanalyticalreagent with at least one vesicle, generated from a living cell,according to claim 113, wherein the one or more receptors are present ina modified form in said one or more vesicles as a component of thebioanalytical reagent.
 121. A bioanalytical reagent with at least onevesicle, generated from a living cell, according to claim 113, whereinthe one or more receptors are present in a form resulting from arecombinant fabrication process in said one or more vesicles as acomponent of the bioanalytical reagent.
 122. A bioanalytical reagentwith at least one vesicle, generated from a living cell, according toclaim 113, comprising the preservation of a binding capability of saidone or more receptors to a specific ligand, this binding capacity beingpresent in said vesicle-generating cell and the receptor beingassociated with the vesicle as a component of the bioanalytical reagent.123. A bioanalytical reagent with at least one vesicle, generated from aliving cell, according to claim 113, wherein the one or more receptorsare selected from the group of signal-transducing receptors that isformed by plasma membrane receptors, such as ion channel receptors, Gprotein-coupled receptors (GPCR), orphan receptors, enzyme-coupledreceptors, such as receptors with an intrinsic tyrosine kinase activity,receptors with an intrinsic serine/threonine kinase activity, furtheronby receptors for growth factors (peptide hormone receptors), receptorsfor chemotactic substances, such as the class of chemokine receptors,and by intracellular hormone receptors, such as steroid hormonereceptors.
 124. A bioanalytical reagent with at least one vesicle,generated from a living cell, according to claim 113, wherein said oneor more vesicles comprise, besides said one or more receptors, furtherbiological compounds (components) from the group that is formed, e.g.,by G-proteins and G-protein regulators (e.g. rasGAP), enzymes, such asadenylate cyclases, phospholipases forming intracellular secondarymessenger compounds (e.g. cAMP (cyclic adenosine monophosphate), cGMP(cyclic guanosine monophosphate), diacyl glycerol (DAG) or inositoltriphosphate (IP3)), enzymes, such as serine, threonine and tyrosinekinases, and tyrosine phosphatases that activate or inhibit proteins byphosphorylation or de-phosphorylation.
 125. A bioanalytical reagent withat least one vesicle, generated from a living cell, according to claim113, wherein biological, biochemical or synthetic compounds like cellsurface proteins or cell surface sugars are associated with the outermembrane of the one or more vesicles, which associated compounds areused for the transport of said vesicle to pre-determined destinations,such as cells and/or organs and/or pre-determined tissue in a livingorganism, and/or for the binding to a biological or biochemical orsynthetic recognition element, which specifically recognizes and bindssaid biological or biochemical or synthetic recognition element.
 126. Abioanalytical reagent with at least one vesicle, generated from a livingcell, according to claim 113, wherein the mechanism of signaltransduction comprises a mechanism from among the group of mechanismsthat is formed e.g. from ion conducting, G-protein coupling, activationor inhibition of intra-vesicular ion channels, intra-vesicular releaseof calcium, protein activation or inhibition by enzymaticphosphorylation or de-phosphorylation (kinase cascades; phosphatases),and release or enzymatic formation of secondary messenger compounds,such as cAMP, cGMP or diacyl glycerol (DAG), inositol triphosphate(IP3).
 127. A bioanalytical reagent with at least one vesicle, generatedfrom a living cell, according to claim 113, wherein said mechanism ofsignal transduction comprises a (secondary) functional response of theone or more vesicle-associated receptors after a primary specificinteraction of said one or more receptors with one or more naturaland/or synthetic ligands contained in a sample that is brought intocontact with said vesicle.
 128. A bioanalytical reagent with at leastone vesicle, generated from a living cell, according to claim 113,wherein said mechanism of signal transduction comprises the activationof an ion channel of a receptor associated with a vesicle, as acomponent of said bioanalytical reagent.
 129. A bioanalytical reagentwith at least one vesicle, generated from a living cell, according toclaim 113, wherein said mechanism of signal transduction comprises thebinding of a G-protein to a receptor associated with a vesicle, as acomponent of said bioanalytical reagent.
 130. A bioanalytical reagentwith at least one vesicle, generated from a living cell, according toclaim 113, wherein said mechanism of signal transduction comprises theinternal release of ions, such as Ca²⁺, or of other messenger compounds,such as cAMP or cGMP.
 131. A bioanalytical reagent with at least onevesicle, generated from a living cell, according to claim 113, whereinsaid mechanism of signal transduction comprises the enzymaticdecomposition of a substrate to a product by a vesicle-associatedenzyme.
 132. A bioanalytical reagent with at least one vesicle,generated from a living cell, according to claim 127, wherein a(secondary) functional response as part of said mechanism of signaltransduction occurs after interaction between one or more natural and/orsynthetic ligands or co-factors contained in a sample brought intocontact with said vesicle on the one hand and naturally or recombinantlygenerated proteins associated with said vesicle on the other.
 133. Abioanalytical reagent with at least one vesicle, generated from a livingcell, according to claim 113, wherein said one or more vesiclesadditionally comprise components for generation of an experimentallydetectable signal.
 134. A bioanalytical reagent with at least onevesicle, generated from a living cell, according to claim 133, whereinsaid additional components for generation of an experimentallydetectable signal are associated with the further biological compounds(components) according to claim 128, these further biological compounds(components) being associated with the one or more vesicles as acomponent of said bioanalytical reagent.
 135. A bioanalytical reagentwith at least one vesicle, generated from a living cell, according toclaim 133, wherein said additional components for generation of anexperimentally detectable signal are associated with a receptor.
 136. Abioanalytical reagent with at least one vesicle, generated from a livingcell, according to claim 133, wherein said additional components forgeneration of an experimentally detectable signal are parts of fusionproteins that are associated with said one or more vesicles.
 137. Abioanalytical reagent with at least one vesicle, generated from a livingcell, according to claim 133, wherein said additional components forgeneration of an experimentally detectable signal are selected from thegroup of components formed by absorptive indicators and luminescentindicators, luminescence labels, luminescent nanoparticles, absorptiveindicator proteins and luminescent indicator proteins, such as BFP(“blue fluorescent protein”), GFP (“green fluorescent protein”) or RFP(“red fluorescent protein”), artificial luminescent amino acids,radioactive labels, spin labels, such as NMR labels or ESR labels, ionindicators, especially pH and calcium indicators, or potential-dependentindicators, such as potential-dependent luminescence labels, or redoxcomplexes.
 138. A bioanalytical reagent with at least one vesicle,generated from a living cell, according to claim 133, wherein saidadditional components for generation of an experimentally detectablesignal are generated from the same cell from which the vesicle wasgenerated.
 139. A bioanalytical reagent with at least one vesicle,generated from a living cell, according to claim 133, wherein saidadditional components for generation of an experimentally detectablesignal are inserted into the vesicle after its formation.
 140. Abioanalytical reagent with at least one vesicle, generated from a livingcell, according to claim 113, wherein the functionality of a receptorassociated with a vesicle being a component of said bioanalyticalreagent is preserved upon storage under deep-frozen conditions for aperiod of at least one week, preferably of at least one month,especially preferred for at least one year.
 141. A bioanalytical reagentwith at least one vesicle, generated from a living cell, according toclaim 113, wherein it is characterized by a shelf life of at least oneweek under sterile conditions in cooled buffer solution, i.e. at atemperature below ambient temperature, e.g. at 4° C.
 142. A method forproduction of a bioanalytical reagent with a vesicle generated from aliving cell, according to claim 113, wherein said vesicle was producedfrom a living cell comprising at least one receptor, and wherein amechanism of signal transduction triggered by said receptor in saidliving cell is preserved in said vesicle as a component of thebioanalytical reagent.
 143. A method for production of a bioanalyticalreagent with a vesicle generated from a living cell, according to claim142, wherein the constriction of said vesicle from said living cell iseffected after application of cytochalasin B and/or cytochalasin D. 144.A method according to claim 142, wherein said method comprises theapplication of shear forces and/or of centrifugation steps, for exampleupon exposure to a gradient of sucrose, and/or the application ofchromatographic steps, for example by separation into fractions ofdifferent size distributions, and/or the application of filtration stepsand/or the application of electrophoretic methods.
 145. A methodaccording to claim 142, wherein the interior of a vesicle produced bysaid method is free from cell nucleus material, so that replicativeprocesses do not occur.
 146. A method according to claim 142, comprisingthe preservation of a binding capability of said one or more receptorsto a specific ligand, this binding capability being present in saidvesicle-generating cell and the receptor being associated with thevesicle as a component of the bioanalytical reagent.
 147. A methodaccording to claim 142, wherein the one or more receptors are selectedfrom the group of signal-transducing receptors that is formed by plasmamembrane receptors, such as ion channel receptors, G protein-coupledreceptors (GPCR), orphan receptors, enzyme-coupled receptors, such asreceptors with an intrinsic tyrosine kinase activity, receptors with anintrinsic serine/threonine kinase activity, furtheron by receptors forgrowth factors (peptide hormone receptors), receptors for chemotacticsubstances, such as the class of chemokine receptors, and byintracellular hormone receptors, such as steroid hormone receptors. 148.A method according to claim 142, wherein said one or more vesiclesproduced by this method comprise, besides said one or more receptors,further biological compounds (components) from the group that is formede.g. by G-proteins and G-protein regulators (e.g. rasGAP), enzymes, suchas adenylate cyclases, phospholipases forming intracellular secondarymessenger compounds (e.g. cAMP (cyclic adenosine monophosphate), cGMP(cyclic guanosine monophosphate), diacyl glycerol (DAG) or inositoltriphosphate (IP3)), enzymes, such as serine, threonine, and tyrosinekinases, and tyrosine phosphatases that activate or inhibit proteins byphosphorylation or de-phosphorylation.
 149. A method according to claim142, wherein biological, biochemical or synthetic compounds, such ascell surface proteins or cell surface sugars, are associated with theouter membrane of the one or more vesicles produced by this method,these compounds being used for the transport of said vesicle topre-determined destinations, such as cells and/or organs and/orpre-determined tissue in a living organism, and/or for the binding to abiological or biochemical or synthetic recognition element, whichspecifically recognizes and binds said biological or biochemical orsynthetic recognition element.
 150. A method according to claim 142,wherein said one or more vesicles produced by this method additionallycomprise components for generation of an experimentally detectablesignal.
 151. A method for production of a bioanalytical reagent with avesicle generated from a living cell, according to claim 142, whereinsaid vesicle is merged with an artificial lipid vesicle to form a mixedvesicle.
 152. A method according to claim 151, wherein said mixedvesicle is substantially enlarged in comparison to the vesicle generatedfrom a living cell, for example to a diameter of 5 μm-50 μm.
 153. Amethod according to claim 151, wherein said mixed vesicle comprisesadditional natural and/or artificial lipids and/or also additionalproteins with additional functionalities, in comparison to the vesiclegenerated from a living cell.
 154. A bioanalytical detection method witha bioanalytical reagent according to claim 113, wherein said detectionmethod is selected from the group that is formed, for example, byoptical detection methods, such as refractrometric methods, surfaceplasmon resonance, optical absorption measurements (e.g. internalreflection methods using a highly refractive material, in combinationwith infrared spectroscopic measurements) or luminescence detection(e.g. fluorescence correlation spectroscopy), detection of energy orcharge transfer, mass spectroscopy, electrical or electrochemicaldetection methods, such as electrophysiology, patch clamp techniques,impedance measurements, electronic resonance measurements, such aselectron spin resonance or nuclear spin resonance, gravimetric methods(e.g. electrical crystal balance measurements), radioactive methods, orby electrophoretic measurements.
 155. A bioanalytical detection methodaccording to claim 154, wherein said method is performed in homogeneoussolution.
 156. A bioanalytical detection method according to claim 154,wherein said method is performed using a measurement arrangement with atleast 2 electrodes and separate compartments adequate for receivingliquids, wherein a solid carrier (preferably as an electricallyisolating separation wall), comprising at least one aperture andseparating at least 2 compartments, is located between two electrodesfacing each other, the electrodes being of any geometrical form and eachextending into at least one compartment or being in contact with atleast one compartment.
 157. A bioanalytical detection method accordingto claim 156, wherein said measurement arrangement is provided withmeans on one side or on both sides of the carrier which enable a supplyof liquid and/or a storage of liquid and/or an exchange of liquid and/orthe addition of vesicles generated from a living cell, from abioanalytical reagent according to the invention, between carrier andelectrode.
 158. A bioanalytical detection method according to claim 156,wherein the one or more apertures of said measurement arrangement havesuch a diameter that, in the presence of a potential difference over themeasurement arrangement and mediated by the two or more electrodes, aninhomogeneous electrical field is generated around the aperture, saidfield having an increasing value with decreasing distance from theaperture and said field being capable of moving vesicleselectrophoretically towards the aperture, said vesicles being locatedclose to said aperture and generated from a living cell, from abioanalytical reagent according to the invention.
 159. A bioanalyticaldetection method according to claim 156, wherein the one or moreapertures of said measurement arrangement have such a diameter thatvesicles generated from a living cell, from a bioanalytical reagentaccording to the invention, can be positioned over or within theaperture by means of a hydrodynamic or electrokinetic flow or by othermechanical manipulation (e.g. by means of optical tweezers, forcemicroscope or by a micro manipulator).
 160. A bioanalytical detectionmethod according to claim 156, wherein the carrier of said measurementarrangement is provided with an electrically charged surface whichexerts attractive force on vesicles generated from a living cell, from abioanalytical reagent according to the invention, or is provided with anadhesion promoting layer for binding said vesicles on its surface. 161.A bioanalytical detection method according to claim 156, whereinvesicles generated from a living cell, from a bioanalytical reagentaccording to the invention, are inserted between separation wall orcarrier and electrode into a compartment filled or not filled withbuffer beforehand, and wherein said vesicles are moved towards theaperture by means of an electrical potential difference applied to theelectrodes, or are positioned on the aperture by hydrodynamic orelectrokinetic flow and/or are positioned on the aperture mechanically(e.g. by means of optical tweezers, force microscope or by a micromanipulator).
 162. A bioanalytical detection method according to claim156, wherein vesicles generated from a living cell, from a bioanalyticalreagent according to the invention, are positioned on said aperture, thevesicle membranes form an electrically close contact with the carrierover the aperture, and a measurement of the (electrical) membraneresistance is enabled.
 163. A bioanalytical detection method accordingto claim 156, wherein artificial lipid vesicles with a diameter largerthan the diameter of said aperture are added to at least onecompartment, in order to generate a planar lipid bilayer on the surfaceof the carrier and extending over the aperture, and wherein thenvesicles generated from a living cell, from a bioanalytical reagentaccording to the invention are added to said compartment, in order tomerge said vesicles with the generated lipid membrane and to makereceptors that are associated with said vesicles generated from livingcells accessible for electrical or optical measurements.
 164. Abioanalytical detection method according to claim 156, wherein membraneproteins are inserted into a vesicle generated from a living cell, afterpositioning said vesicle on an aperture.
 165. A bioanalytical detectionmethod according to claim 156, wherein a vesicle generated from a livingcell located over an aperture or a planar membrane generated from saidvesicle and spanning an aperture is accessible for optical measurements,especially for fluorescence measurements, or for simultaneous opticaland electrical measurements, to which it is subjected.
 166. Abioanalytical detection method according to claim 156, wherein ameasurement arrangement or a measurement system with several apertureson one carrier is used, and wherein measurements on at least twoapertures are performed sequentially and/or in parallel.
 167. Abioanalytical detection method according to claim 156, wherein amultitude of vesicles generated from living cells, from a bioanalyticalreagent according to the invention, is arranged in an array on a solid,electrically isolating carrier, wherein said array of vesicles isbrought into electrically isolating contact with an array of patch-clamppipets in a geometrical arrangement similar to that of the vesiclearray, in order to enable a simultaneous performance of electricalmeasurements independently of each other or simultaneous electrical andoptical measurements on a multitude of individual vesicles.
 168. Abioanalytical detection method according to claim 154, wherein the atleast one vesicle generated from a living cell, comprising at least onereceptor, from a bioanalytical reagent according to the invention, isimmobilized on the surface of a solid support.
 169. A bioanalyticaldetection method according to claim 168, wherein a mechanism of a signaltransduction triggered by said receptor in said living cell is retainedin a vesicle generated from the cell after immobilization of thevesicle.
 170. A bioanalytical detection method with at least one vesicleimmobilized on the surface of a solid support, the vesicle beinggenerated from a living cell, from a bioanalytical reagent according toclaim 113, comprising at least one receptor, wherein a mechanism of asignal transduction triggered by said receptor in said living cell ispreserved in a vesicle generated from the cell after immobilization ofthe vesicle.
 171. A bioanalytical detection method according to claim170, wherein vesicles, each comprising at least one receptor, areimmobilized in discrete measurement areas (d) with one or more vesicleseach on the surface of said solid support.
 172. A bioanalyticaldetection method according to claim 170, wherein vesicles with at leasttwo different kinds of receptor are immobilized in a multitude ofmeasurement areas (d), wherein preferably within an individualmeasurement area always vesicles with a uniform kind of receptor areimmobilized.
 173. A bioanalytical detection method according to claim168, wherein the immobilization of the one or more vesicles generatedfrom a living cell, on the surface of said solid support, is performedupon covalent binding or upon physical adsorption (electrostatic orvan-der-Waals interaction or hydrophilic or hydrophobic interaction or acombination of these interactions).
 174. A bioanalytical detectionmethod according to claim 167, wherein an adhesion-promoting layer isdeposited between the surface of said solid support and the one or morevesicles immobilized thereon.
 175. A bioanalytical detection methodaccording to claim 174, wherein the adhesion-promoting layer comprises achemical compound of the group of silanes, epoxides, functionalized,charged or polar polymers and “self-organized functionalized mono ormultiple layers”.
 176. A bioanalytical detection method according toclaim 174, wherein the adhesion-promoting layer comprises amonomolecular layer of mainly one kind of protein, such as serumalbumins or streptavidin, or of modified proteins, such as biotinylatedserum albumin.
 177. A bioanalytical detection method according to claim174, wherein the adhesion-promoting layer comprises self-organizedalkane-terminated monolayers of mainly one kind of chemical orbiochemical molecules.
 178. A bioanalytical detection method accordingto claim 174, comprising association with the adhesion-promoting layerof biological or biochemical or synthetic recognition elements whichrecognize and bind a vesicle generated from a living cell withsurface-associated biological or biochemical or synthetic components forspecific recognition and binding from the bioanalytical reagent, whereinbiological, biochemical or synthetic compounds like cell surfaceproteins or cell surface sugars are associated with the outer membraneof the one or more vesicles, which associated compounds are used for thetransport of said vesicle to pre-determined destinations, such as cellsand/or organs and/or pre-determined tissue in a living organism, and/orfor the binding to a biological or biochemical or synthetic recognitionelement, which specifically recognizes and binds said biological orbiochemical or synthetic recognition element.
 179. A bioanalyticaldetection method according to claim 154, wherein at least one ligand fora receptor, which is bound to a vesicle generated from a living cell,from a bioanalytical reagent according to the invention, is immobilized,optionally by means of a spacer molecule, on the surface of the solidsupport.
 180. A bioanalytical detection method according to claim 179,wherein at least two different ligands for receptors, which are bound toa vesicle generated from a living cell, from a bioanalytical reagentaccording to the invention, are immobilized in a multitude ofmeasurement areas (d), wherein preferably a uniform kind of ligand isimmobilized within an individual measurement area.
 181. A bioanalyticaldetection method according to claim 179, wherein said ligands areimmobilized on the surface of the solid support upon covalent binding orupon physical adsorption (e.g. electrostatic or van-der-Waalsinteraction or hydrophilic or hydrophobic interaction or a combinationof these interactions).
 182. A bioanalytical detection method accordingto claim 179, wherein an adhesion-promoting layer is applied between thesurface of the solid support and said ligands immobilized thereon. 183.A bioanalytical detection method according to claim 171, wherein regionsbetween the laterally separated measurement areas, with vesicles,generated from living cells (from a bioanalytical reagent according tothe invention) immobilized in these measurement areas, or with ligandsfor receptors that are bound to vesicles generated from living cells(from a bioanalytical reagent according to the invention), and/orregions within these measurement areas, between the compoundsimmobilized therein, are “passivated” in order to minimize non-specificbinding of analytes or of their detection reagents, i.e., that compoundswhich are “chemically neutral” towards the analyte are deposited betweenthe laterally separated measurement areas (d) and/or within thesemeasurement areas (d) between said immobilized compounds, the“chemically neutral” compounds preferably being composed of the groupsthat are formed by albumins, casein, detergents, such as Tween 20,detergent/lipid mixtures (of synthetic and/or natural lipids), syntheticand natural lipids or also hydrophilic polymers, such as polyethyleneglycols or dextrans.
 184. A bioanalytical detection method according toclaim 156, wherein the material of the solid support (carrier) withimmobilized vesicles, generated from living cells (from a bioanalyticalreagent according to the invention), or with immobilized ligands forreceptors that are bound to vesicles generated from living cells (from abioanalytical reagent according to the invention), comprises a materialof the group which is formed e.g. by moldable, sprayable or millableplastics, carbon compounds, metals, such as gold, silver, copper, metaloxides or silicates, such as glass, quartz or ceramics, or silicon orgermanium or ZnSe or a mixture of these materials.
 185. A bioanalyticaldetection method according to claim 156, wherein the surface of saidsolid support (carrier) is essentially planar.
 186. A bioanalyticaldetection method according to claim 156, wherein said solid support(carrier) is an optical or electronic sensor platform.
 187. Abioanalytical detection method according to claim 156, wherein saidsolid support (carrier) is transparent at least in a region ofwavelengths in the ultraviolet to infrared spectrum and comprisespreferably a material from the group that is formed e.g. by moldable,sprayable or millable plastics, carbon compounds, metals, metal oxidesor silicates, such as glass, quartz or ceramics, or silicon or germaniumor ZnSe or a mixture of these materials.
 188. A bioanalytical detectionmethod according to claim 186, wherein said solid support is an opticalwaveguide used as a sensor platform.
 189. A bioanalytical detectionmethod according to claim 186, wherein said solid support is an opticalthin-film waveguide used as a sensor platform, with an initial opticallytransparent layer (a) with refractive index n1 on a second opticallytransparent layer (b) with refractive index n₂, wherein n₂, wherein n,>n₂.
 190. A bioanalytical detection method according to claim 188,wherein the sensor platform as a solid support is divided into two ormore discrete waveguiding regions.
 191. A bioanalytical detection methodaccording to claim 189, wherein the material of the second opticallytransparent layer (b) of the sensor platform as a solid support isselected from the group that is formed by silicates, such as glass orquartz, or transparent moldable, sprayable or millable, especiallythermoplastic plastics, such as polycarbonates, polyimides, polymethylmethacrylates, or polystyrenes.
 192. A bioanalytical detection methodaccording to claim 189, wherein the refractive index of the firstoptically transparent layer (a) of the sensor platform as a solidsupport is greater than 1.8.
 193. A bioanalytical detection methodaccording to claim 189, wherein the first optically transparent layer(a) comprises a material of the group of TiO₂, ZnO, Nb₂O₅, Ta₂O₅, HfO₂,or ZrO₂, preferably of TiO₂ or Ta₂O₅ or Nb₂O₅.
 194. A bioanalyticaldetection method according to claim 189, wherein an additional opticallytransparent layer (b′) with lower refractive index than layer (a) andwith a thickness of 5 nm-10 000 nm, preferably of 10 nm-1000 nm, islocated between the optically transparent layers (a) and (b) and incontact with layer (a).
 195. A bioanalytical detection method accordingto claim 189, wherein the in-coupling of excitation light into theoptically transparent layer (a) to the measurement areas (d) on thesensor platform as a solid support is performed using one or moreoptical in-coupling elements from the group formed by prism couplers,evanescent couplers comprising joined optical waveguides withoverlapping evanescent fields, butt-couplers with focusing lenses,preferably cylindrical lenses, arranged in front of a front face (distalend) of the waveguiding layer, and grating couplers.
 196. Abioanalytical detection method according to claim 189, wherein thein-coupling of excitation light into the optically transparent layer (a)to the measurement areas (d) is performed using one or more gratingstructures (c) that are formed in the optically transparent layer (a).197. A bioanalytical detection method according to claim 156, whereinone or more liquid samples, comprising vesicles generated from livingcells (from a bioanalytical reagent according the invention), withassociated receptors, are brought into contact with the ligands forthese receptors, immobilized in one or more measurement areas, andwherein a signal change caused by a binding of the receptors associatedwith said vesicles to their immobilized ligands is measured.
 198. Abioanalytical detection method according to claim 197, wherein thesignal transduction of receptors associated with vesicles generated fromliving cells (from a bioanalytical reagent according to the invention)after binding of these receptors to their immobilized ligands, ismeasured, wherein this signal transduction can be triggered, forexample, by binding of further ligands to the receptors associated withsaid vesicles, or by other inducing influences.
 199. A bioanalyticaldetection method according to claim 197, wherein the binding ofreceptors that are associated with vesicles generated from living cells(from a bioanalytical reagent according to the invention) to saidimmobilized ligands occurs in competition with the binding of thesereceptors associated with said vesicles to ligands in free solution.200. A bioanalytical detection method according to claim 156, whereinone or more liquid samples are brought into contact with the vesicles,which are generated from living cells (from a bioanalytical reagentaccording to the invention) and immobilized in one or more measurementareas, along with their associated receptors, and wherein a signalchange resulting from the binding of ligands to said receptors or fromother inducing influences on said receptors is measured.
 201. Abioanalytical detection method according to claim 156, wherein one ormore liquid samples are brought into contact with the vesicles, whichare generated from living cells (from a bioanalytical reagent accordingto the invention) and immobilized in one or more measurement areas,along with their associated receptors, and wherein the signaltransduction of those receptors resulting from the binding of ligands tosaid receptors or from other inducing influences on said receptors ismeasured.
 202. A bioanalytical detection method according to claim 200,wherein the binding of ligands from a supplied sample to receptors thatare associated with the immobilized vesicles generated from living cells(from a bioanalytical reagent according to the invention) occurs incompetition with the binding of these ligands to receptors in freesolution which are optionally associated with vesicles.
 203. Abioanalytical detection method according to claim 197, wherein one ormore liquid samples, comprising vesicles generated from living cells(from a bioanalytical reagent according to the invention) withassociated receptors, are brought into contact with the ligands forthese receptors, the ligands being immobilized in one or moremeasurement areas, excitation light from one or more light sources ofsimilar or different wavelengths is in-coupled to the measurement areas(d) by one or more grating structures (c), and the change of opticalsignals emanating from one or more measurement areas (d), caused by abinding of the receptors associated with said vesicles to theirimmobilized ligands, is measured.
 204. A bioanalytical detection methodaccording to claim 200, wherein one or more liquid samples are broughtinto contact with the vesicles immobilized in one or more measurementareas (d), along with their associated receptors, excitation light fromone or more light sources of similar or different wavelengths isin-coupled to the measurement areas (d) by one or more gratingstructures (c), and the change of optical signals emanating from one ormore measurement areas (d), caused by the binding of the ligands to saidreceptors or by other inducing influences on said receptors, ismeasured.
 205. A bioanalytical detection method according to claim 204,wherein said changes of optical signals from the measurement areas (d)are caused by changes of the effective refractive index in thenear-field of the optically transparent layer (a) in these measurementareas and are measured at the actual excitation wavelength.
 206. Abioanalytical detection method according to claim 204, wherein saidchanges of optical signals from the measurement areas (d) are changes ofone or more luminescences of similar or different wavelength, which havebeen excited in said measurement areas in the near-field of theoptically transparent layer (a), and which are measured each at awavelength different from the corresponding excitation wavelength. 207.A bioanalytical detection method according to claim 204, wherein the oneor more luminescences and/or measurements of light signals at theexcitation wavelength are determined polarization-selectively, whereinpreferably the one or more luminescences are measured at a polarizationthat is different from the polarization of the excitation light.
 208. Abioanalytical detection method according to claim 154, for thesimultaneous or sequential, quantitative and/or qualitativedetermination of one or more analytes from the group of receptors orligands, chelators or “histidine tag components”, enzymes, enzymeco-factors or inhibitors.
 209. A bioanalytical detection methodaccording to claim 154, wherein the samples to be examined are, forexample, aqueous solutions or surface water or soil or plant extracts orbio- or process broths, or are taken from biological tissue fractions orfrom food, or odorous or flavoring substances or cosmetic compounds.210. The use of a vesicle as a component of a bioanalytical reagentaccording to claim 113 for the enrichment of membrane receptors or forthe enrichment of proteins (such as antigens) triggering animmunological response in a two- or three-dimensional phase, which canthen e.g. be administered to living organisms (e.g. to stimulate immunedefense processes).
 211. The use of a vesicle, as a component of abioanalytical reagent according to claim 113 as a compartment fortherapeutic, diagnostic, photosensitive or other biologically activecompounds for administration to a living organism.
 212. The use of abioanalytical reagent according to claim 113 for investigatingreceptor-ligand interactions, especially for determining the bindingstrength and kinetic parameters of these interactions between a receptorand its ligand, or for determining the channel activity of an ionchannel receptor after ligand binding or other inducing influences onsaid receptor, or for determining the enzymatic activity of enzymesassociated with a vesicle, as a component of a bioanalytical reagentaccording to the invention, or for determining secondary messengercompounds after ligand binding to a receptor resulting in a signaltransduction, or for determining protein-protein interactions, or fordetermining protein kinases.
 213. The use of a bioanalytical reagentaccording to claim 113 for quantitative and/or qualitative analyses fordetermining chemical, biochemical or biological analytes in screeningmethods in pharmaceutical research, combinatorial chemistry, clinicaland pre-clinical development, for real-time binding studies and fordetermining kinetic parameters in affinity screening and in research,for qualitative and quantitative analyte determinations, especially forDNA- and RNA analytics, for generation of toxicity studies and for thedetermination of expression profiles, and for determining antibodies,antigens, pathogens or bacteria in pharmaceutical product developmentand research, human and veterinary diagnostics, agrochemical productdevelopment and research, for symptomatic and pre-symptomatic plantdiagnostics, for patient stratification in pharmaceutical productdevelopment and for therapeutic drug selection, for determiningpathogens, nocuous agents and germs, especially of salmonella, prionsand bacteria, in food and environmental analytics, and for analysis andquality control of odorous and flavoring substances.
 214. The use of abioanalytical detection method according to claim 154 for investigatingreceptor-ligand interactions, especially for determining the bindingstrength and kinetic parameters of these interactions between a receptorand its ligand, or for determining the channel activity of an ionchannel receptor after ligand binding or other inducing influences onsaid receptor, or for determining the enzymatic activity of enzymesassociated with a vesicle, as a component of a bioanalytical reagentaccording to the invention, or for determining secondary messengercompounds after ligand binding to a receptor resulting in a signaltransduction, or for determining protein-protein interactions, or fordetermining protein kinases.
 215. The use of a bioanalytical detectionmethod according to claim 154 for quantitative and/or qualitativeanalyses for determining chemical, biochemical or biological analytes inscreening methods in pharmaceutical research, combinatorial chemistry,clinical and pre-clinical development, for real-time binding studies andfor determining kinetic parameters in affinity screening and inresearch, for qualitative and quantitative analyte determinations,especially for DNA- and RNA analytics, for generation of toxicitystudies and for the determination of expression profiles, and fordetermining antibodies, antigens, pathogens or bacteria inpharmaceutical product development and research, human and veterinarydiagnostics, agrochemical product development and research, forsymptomatic and pre-symptomatic plant diagnostics, for patientstratification in pharmaceutical product development and for therapeuticdrug selection, for determining pathogens, nocuous agents and germs,especially of salmonella, prions and bacteria, in food and environmentalanalytics, and for analysis and quality control of odorous and flavoringsubstances.